https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Diana+TrautmannProteopedia - User contributions [en]2026-09-23T11:54:15ZUser contributionsMediaWiki 1.43.8https://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1230357Sandbox Reserved 1972011-04-14T17:21:51Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation [http://www.uta.edu/faculty/sawasthi/Enzymology-4351-5324/Class%20Syllabus%20Enzymology/ribonucleaseA.pdf]. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is performed by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and is then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize for his work on protein folding, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. The Anfinsen experiment has ignited the interest in protein folding. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Proline Conformation' scene='Sandbox_Reserved_197/Rnase_a_wild_type/7' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue [http://www.ncbi.nlm.nih.gov/pubmed/9605332]. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/3'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop [http://onlinelibrary.wiley.com/doi/10.1110/ps.051610505/full]. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Disulfide bonds are shown in yellow' scene='Sandbox_Reserved_197/Rnase_a_wild_type/8' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/6'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/6'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/8'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/7'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/8'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, <scene name='Sandbox_Reserved_197/Residues_34-45/1'>34-45</scene> and <scene name='Sandbox_Reserved_197/Residues_83-101/1'>83-101</scene>, where there are increased levels of disorder and a destabilized hydrophobic core [http://www.ncbi.nlm.nih.gov/pubmed/9605332]. <br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/2'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/3'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1230355Sandbox Reserved 1972011-04-14T17:20:49Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
=''Ribonuclease A and Protein Folding''=<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation [http://www.uta.edu/faculty/sawasthi/Enzymology-4351-5324/Class%20Syllabus%20Enzymology/ribonucleaseA.pdf]. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is performed by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and is then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize for his work on protein folding, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. The Anfinsen experiment has ignited the interest in protein folding. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Proline Conformation' scene='Sandbox_Reserved_197/Rnase_a_wild_type/7' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue [http://www.ncbi.nlm.nih.gov/pubmed/9605332]. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/3'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop [http://onlinelibrary.wiley.com/doi/10.1110/ps.051610505/full]. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Disulfide bonds are shown in yellow' scene='Sandbox_Reserved_197/Rnase_a_wild_type/8' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/6'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/6'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/8'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/7'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/8'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, <scene name='Sandbox_Reserved_197/Residues_34-45/1'>34-45</scene> and <scene name='Sandbox_Reserved_197/Residues_83-101/1'>83-101</scene>, where there are increased levels of disorder and a destabilized hydrophobic core [http://www.ncbi.nlm.nih.gov/pubmed/9605332]. <br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/2'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/3'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1230354Sandbox Reserved 1972011-04-14T17:18:59Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation [http://www.uta.edu/faculty/sawasthi/Enzymology-4351-5324/Class%20Syllabus%20Enzymology/ribonucleaseA.pdf]. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is performed by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and is then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize for his work on protein folding, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. The Anfinsen experiment has ignited the interest in protein folding. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Proline Conformation' scene='Sandbox_Reserved_197/Rnase_a_wild_type/7' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue [http://www.ncbi.nlm.nih.gov/pubmed/9605332]. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/3'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop [http://onlinelibrary.wiley.com/doi/10.1110/ps.051610505/full]. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Disulfide bonds are shown in yellow' scene='Sandbox_Reserved_197/Rnase_a_wild_type/8' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/6'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/6'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/8'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/7'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/8'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, <scene name='Sandbox_Reserved_197/Residues_34-45/1'>34-45</scene> and <scene name='Sandbox_Reserved_197/Residues_83-101/1'>83-101</scene>, where there are increased levels of disorder and a destabilized hydrophobic core [http://www.ncbi.nlm.nih.gov/pubmed/9605332]. <br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/2'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/3'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1230353Sandbox Reserved 1972011-04-14T17:08:17Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is performed by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and is then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize for his work on protein folding, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. The Anfinsen experiment has ignited the interest in protein folding. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Proline Conformation' scene='Sandbox_Reserved_197/Rnase_a_wild_type/7' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/3'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Disulfide bonds are shown in yellow' scene='Sandbox_Reserved_197/Rnase_a_wild_type/8' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/6'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/6'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/8'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/7'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/8'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, <scene name='Sandbox_Reserved_197/Residues_34-45/1'>34-45</scene> and <scene name='Sandbox_Reserved_197/Residues_83-101/1'>83-101</scene>, where there are increased levels of disorder and a destabilized hydrophobic core. <br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/2'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/3'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1230352Sandbox Reserved 1972011-04-14T17:03:49Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is performed by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and is then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize for his work on protein folding, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. The Anfinsen experiment has ignited the interest in protein folding. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Proline Conformation' scene='Sandbox_Reserved_197/Rnase_a_wild_type/7' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/3'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Disulfide bonds are shown in yellow' scene='Sandbox_Reserved_197/Rnase_a_wild_type/8' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/6'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/6'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/7'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/7'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/8'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, <scene name='Sandbox_Reserved_197/Residues_34-45/1'>34-45</scene> and <scene name='Sandbox_Reserved_197/Residues_83-101/1'>83-101</scene>, where there are increased levels of disorder and a destabilized hydrophobic core. <br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/2'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/3'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1230231Sandbox Reserved 1972011-04-13T22:16:21Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is performed by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and is then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize for his work on protein folding, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. The Anfinsen experiment has ignited the interest in protein folding. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Proline Conformation' scene='Sandbox_Reserved_197/Rnase_a_wild_type/7' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/3'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Disulfide bonds are shown in yellow' scene='Sandbox_Reserved_197/Rnase_a_wild_type/8' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/5'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/5'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/5'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/6'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/7'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, 34-45 and 83-101, where there are increased levels of disorder and a destabilized hydrophobic core. <br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/2'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1230230Sandbox Reserved 1972011-04-13T22:13:18Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is performed by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and is then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize for his work on protein folding, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. The Anfinsen experiment has ignited the interest in protein folding. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/7' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/3'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/8' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/5'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/5'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/5'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/6'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/7'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, 34-45 and 83-101, where there are increased levels of disorder and a destabilized hydrophobic core. <br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/2'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1230229Sandbox Reserved 1972011-04-13T22:00:22Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is performed by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and is then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize for his work on protein folding, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. The Anfinsen experiment has ignited the interest in protein folding. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/7' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/8'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/2'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/2'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/6' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/5'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/5'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/5'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/6'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/7'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, 34-45 and 83-101, where there are increased levels of disorder and a destabilized hydrophobic core. <br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/2'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1230227Sandbox Reserved 1972011-04-13T21:57:39Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is performed by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and is then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize for his work on protein folding, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. The Anfinsen experiment has ignited the interest in protein folding. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/8'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/2'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/2'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/6' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/5'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/5'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/5'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/6'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/7'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, 34-45 and 83-101, where there are increased levels of disorder and a destabilized hydrophobic core. <br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/2'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228139Sandbox Reserved 1972011-04-12T20:18:24Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is done by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acids. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated, based on the structure of RNase A variants, with several mutations to the wild type amino acid sequences. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/2'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/6'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/8'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/2'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/2'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small and flexible and assumes a ''cis'' conformation. In RNase A both Pro93 and Pro114 are involved in β turns. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/6' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/5'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/5'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/5'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/6'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/7'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/2'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228133Sandbox Reserved 1972011-04-12T19:15:09Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and Fast Atom Bombardment Mass Spectrometry [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. FABMS is done by mixing the material to be analyzed (RNase A) with a non-volatile environment called a matrix and then bombarded by high energy molecules within a vacuum. Through this technique it has been found that RNase A is composed of four anti-parallel β-sheets and three α-helixes. Presence of four disulfide bonds and two ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acids. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations were demonstrated, based on the structure of RNase A variants, with several mutations to the wild type amino acid sequences. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/2'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/6'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/8'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding. <br />
<br />
The <scene name='Sandbox_Reserved_197/Cis-proline114/2'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/2'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/5'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/5'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/5'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/6'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/7'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. When a proline residue or disulfide bond is removed from RNase A, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding, because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/2'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228121Sandbox Reserved 1972011-04-12T18:41:58Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The <scene name='Sandbox_Reserved_197/Active_site/2'>active site</scene> lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/2'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/6'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/8'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the protein. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/2'>Asn113-Pro114</scene> resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/2'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop from where it is located. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein causes formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/5'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/5'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/5'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/6'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/7'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a ''cis'' proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228116Sandbox Reserved 1972011-04-12T18:31:58Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The <scene name='Sandbox_Reserved_197/Active_site/2'>active site</scene> lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/2'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/6'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/8'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the protein. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/2'>Asn113-Pro114</scene> resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/2'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop from where it is located. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein causes formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a ''cis'' proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228115Sandbox Reserved 1972011-04-12T18:30:52Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The <scene name='Sandbox_Reserved_197/Active_site/2'>active site</scene> lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/2'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/6'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/8'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the protein. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/2'>Asn113-Pro114</scene> resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/2'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop where it is located. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein causes formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a ''cis'' proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228110Sandbox Reserved 1972011-04-12T18:22:31Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The <scene name='Sandbox_Reserved_197/Active_site/2'>active site</scene> lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/2'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/6'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/8'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the protein. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/2'>Asn113-Pro114</scene> resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop where it is located. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein causes formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a ''cis'' proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228107Sandbox Reserved 1972011-04-12T18:18:30Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The <scene name='Sandbox_Reserved_197/Active_site/2'>active site</scene> lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/2'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/6'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/8'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the protein. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>Asn113-Pro114</scene> resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop where it is located. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein causes formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a ''cis'' proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228103Sandbox Reserved 1972011-04-12T18:13:05Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The <scene name='Sandbox_Reserved_197/Active_site/2'>active site</scene> lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. <br />
Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/2'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/6'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the protein. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>Asn113-Pro114</scene> resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop where it is located. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein causes formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a ''cis'' proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228101Sandbox Reserved 1972011-04-12T18:01:36Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The <scene name='Sandbox_Reserved_197/Active_site/2'>active site</scene> lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. <br />
The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the protein. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>Asn113-Pro114</scene> resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop where it is located. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein causes formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a ''cis'' proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228100Sandbox Reserved 1972011-04-12T17:51:29Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. <br />
The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the protein. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>Asn113-Pro114</scene> resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop where it is located. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein causes formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a ''cis'' proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228099Sandbox Reserved 1972011-04-12T17:49:09Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. <br />
The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the protein. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>Asn113-Pro114</scene> resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop where it is located. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein causes formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a ''cis'' proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1228097Sandbox Reserved 1972011-04-12T17:47:01Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
[[Image:proteopedia 2d2.png|thumb|450px|Structure of RNase A. Disulfide bonds between cysteine residues are shown in red and proline residues are shown in green. Pink regions indicate β-sheets, blue regions indicate α-helixes and tan regions indicate loop structures.]]<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four ''cis'' proline residues greatly effect the structure and folding kinetics of RNase A. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today.<br />
<br />
== '''Protein Folding''' ==<br />
Interatomic interactions are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Proline Conformation'''===<br />
The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. <br />
The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the protein. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>Asn113-Pro114</scene> resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop where it is located. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein causes formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein. <br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. <br />
Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==='''Summary'''===<br />
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a ''cis'' proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.<br />
<br />
=='''Medical Importance'''==<br />
Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. ''Protein Science''. 7:1225-1258.<br />
<br />
Raines, R.T. (1998) Ribonuclease A. ''Chem. Rev.'' 98:1045-1065.<br />
<br />
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the ''cis''-proline to glycine variant (P114G) of ribonuclease A. ''Protein Sci.'' 14:2862-2870.<br />
<br />
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. ''Science'' 125(3250):691-692.<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223217Sandbox Reserved 1972011-03-31T19:40:36Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein [http://en.wikipedia.org/wiki/Protein_folding]. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation [http://en.wikipedia.org/wiki/Cis_configuration]. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223215Sandbox Reserved 1972011-03-31T19:40:05Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein [http://en.wikipedia.org/wiki/Protein_folding]. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation [[Cis configuration]]. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223211Sandbox Reserved 1972011-03-31T19:35:40Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography [http://en.wikipedia.org/wiki/Crystallography] and FABMS [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein [http://en.wikipedia.org/wiki/Protein_folding]. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223208Sandbox Reserved 1972011-03-31T19:33:48Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein [http://en.wikipedia.org/wiki/Protein_folding]. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223205Sandbox Reserved 1972011-03-31T19:32:19Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves due to the [http://en.wikipedia.org/wiki/Hydrophobic_effect]. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223204Sandbox Reserved 1972011-03-31T19:31:34Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves due to the http://en.wikipedia.org/wiki/Hydrophobic_effect. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223203Sandbox Reserved 1972011-03-31T19:29:39Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves due to the hydrophobic effect. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==<br />
<br />
=='''External Links'''==<br />
http://en.wikipedia.org/wiki/Ribonuclease_A</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223197Sandbox Reserved 1972011-03-31T19:23:44Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves due to the hydrophobic effect. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223191Sandbox Reserved 1972011-03-31T19:22:00Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves due to the hydrophobic effect. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223189Sandbox Reserved 1972011-03-31T19:21:38Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves due to the [[hydrophobic effect]]. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> of the interior that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223188Sandbox Reserved 1972011-03-31T19:19:38Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. Each protein contains <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/1'>hydrophobic and hydrophilic residues</scene>. In the case of these aggregates, the mistake exposes "sticky" <scene name='Sandbox_Reserved_197/Hydrophobic/1'>hydrophobic patches</scene> that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223181Sandbox Reserved 1972011-03-31T19:09:51Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance''==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. In the case of these aggregates, the mistake exposes "sticky" hydrophobic patches that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223180Sandbox Reserved 1972011-03-31T19:09:21Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
==''Medical Importance"==<br />
Protein folding, along with its inhibitions, is immensely important to the human. Such diseases as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to the protein. Proteins can form aberrant aggregates when they do not fold correctly. This abnormaility can be fatally toxic to the human nerve cells. During folding, proteins sometimes make a mistake. In the case of these aggregates, the mistake exposes "sticky" hydrophobic patches that can cause several proteins to stick to one another. In the future researchers hope to design drugs that combat this mistake in the protein folding. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent this problem in the future.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223171Sandbox Reserved 1972011-03-31T18:57:12Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/4' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/4'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/4'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/5'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/6'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1223154Sandbox Reserved 1972011-03-31T18:41:36Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<Structure load='7RSA' size='500' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222648Sandbox Reserved 1972011-03-30T20:58:33Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>''cis'' proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222644Sandbox Reserved 1972011-03-30T20:33:49Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222643Sandbox Reserved 1972011-03-30T20:32:41Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
<scene name='Sandbox_Reserved_197/Rnase_a_wild_type/1'>Ribonuclease A</scene> is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, Cys40-Cys95. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222641Sandbox Reserved 1972011-03-30T20:27:22Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
<br />
<jmol><br />
<jmolMenu><br />
<item><br />
<script>Sandbox_Reserved_197/Tyr92-pro93/5</script><br />
<text>Tyr92-Pro93</text><br />
</item><br />
<item><br />
<script>Sandbox_Reserved_197/Tyr92-pro93/5</script><br />
<text>Tyr92-Pro93</text><br />
</item><br />
</jmolMenu><br />
</jmol><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, Cys40-Cys95. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222639Sandbox Reserved 1972011-03-30T20:19:11Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, Cys40-Cys95. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222638Sandbox Reserved 1972011-03-30T20:16:25Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an alpha-helix and a beta sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, Cys40-Cys95. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222637Sandbox Reserved 1972011-03-30T20:16:07Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
[[Ribonuclease A]] is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallography and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active site of a protein is where the actually binding of the protein to its substrate(s) occur(s) and the <scene name='Sandbox_Reserved_197/Active_site/1'>active site</scene> of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an alpha-helix and a beta sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, Cys40-Cys95. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222636Sandbox Reserved 1972011-03-30T20:11:33Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through [[crystallography]] and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active siteof a protein is where the actually binding of the protein to its substrate(s) occur(s) and the active site of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an alpha-helix and a beta sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, Cys40-Cys95. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222635Sandbox Reserved 1972011-03-30T20:10:49Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing [[RNA degradation]]. The structure of RNase A has been determined through crystallization and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active siteof a protein is where the actually binding of the protein to its substrate(s) occur(s) and the active site of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an alpha-helix and a beta sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, Cys40-Cys95. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222634Sandbox Reserved 1972011-03-30T20:09:15Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallization and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active siteof a protein is where the actually binding of the protein to its substrate(s) occur(s) and the active site of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a ''cis'' conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored ''trans'' conformation. This points to the fact that this ''cis'' bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an alpha-helix and a beta sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, Cys40-Cys95. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222633Sandbox Reserved 1972011-03-30T20:07:48Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallization and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active siteof a protein is where the actually binding of the protein to its substrate(s) occur(s) and the active site of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the "cis" conformation and two in the "trans" conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a "cis" conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored "trans" conformation. This points to the fact that this "cis" bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/2'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/2'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/3'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an alpha-helix and a beta sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/4'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, Cys40-Cys95. As you can see in the variant, there are only 3 disulfide bonds present, shown in red.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1222631Sandbox Reserved 1972011-03-30T19:54:18Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallization and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active siteof a protein is where the actually binding of the protein to its substrate(s) occur(s) and the active site of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7RSA' size='500' frame='true' align='right' caption='Insert caption here' scene='Sandbox_Reserved_197/Rnase_a_wild_type/1' /><br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the "cis" conformation and two in the "trans" conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a "cis" conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored "trans" conformation. This points to the fact that this "cis" bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/1'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/1'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/1'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an alpha-helix and a beta sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/3'>[C40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/3'>Cys40-Cys95</scene>. As you can see in the variant, there are only 3 disulfide bonds present, shown in blue.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1220450Sandbox Reserved 1972011-03-29T23:21:36Z<p>Diana Trautmann: </p>
<hr />
<div><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallization and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active siteof a protein is where the actually binding of the protein to its substrate(s) occur(s) and the active site of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<Structure load='7rsa' size='500' frame='true' align='right' caption='Ribonuclease A' scene='Wild Type' /><br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the "cis" conformation and two in the "trans" conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a "cis" conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored "trans" conformation. This points to the fact that this "cis" bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/1'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/1'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/1'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an alpha-helix and a beta sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/3'>[C40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/3'>Cys40-Cys95</scene>. As you can see in the variant, there are only 3 disulfide bonds present, shown in blue.<br />
<br />
=='''References'''==</div>Diana Trautmannhttps://proteopedia.org/index.php?title=Sandbox_Reserved_197&diff=1220448Sandbox Reserved 1972011-03-29T23:20:06Z<p>Diana Trautmann: </p>
<hr />
<div><Structure load='7rsa' size='500' frame='true' align='right' caption='Ribonuclease A' scene='Wild Type' /><!-- PLEASE DO NOT DELETE THIS TEMPLATE --><br />
{{Template:Johnson_CH462_Spring2011}}<br />
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
<br />
== '''Introduction''' ==<br />
Ribonuclease A is an enzyme involved in catalyzing RNA degradation. The structure of RNase A has been determined through crystallization and FABMS. There are several features of its structure that are pertinent to its folding and function and RNase A has been used time and again to illustrate these important features of protein folding. The active siteof a protein is where the actually binding of the protein to its substrate(s) occur(s) and the active site of Rnase A lies within its cleft. Proteins form interactions between different parts of their structure for stabilization. In RNase A, there are eight cysteine residues present that form four disulfide linkages that contribute to the ordered structure as well as the speed of folding of RNase A. Another important aspect to folding of its is the presence of two ''cis'' proline residues. <br />
<br />
== '''Folding''' ==<br />
There are features of every protein that directly or indirectly effect the folding of that protein. Several of these features have been identified in RNase A by the use of mutants of the native form. These mutations and the study of the kinetics and final structure in comparison to the native form show whether that particular feature is involved in the folding of the protein. <br />
<br />
==='''Proline Conformation'''===<br />
One particular feature of RNase A is the presence of ''cis'' proline residues. In nature, most amino acids reside in a ''trans'' conformation. Due to their cyclic structure, prolines are more stable in a ''cis'' conformation. RNase A contains four proline residues, two reside in the "cis" conformation and two in the "trans" conformation. The <scene name='Sandbox_Reserved_197/Tyr92-pro93/5'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a <scene name='Sandbox_Reserved_197/P93a/7'>P93A</scene> mutation, a "cis" conformation still forms; this is an unlikely conformation for an alanine residue. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its favored "trans" conformation. This points to the fact that this "cis" bond formation is a key component to the protein structure of RNase A. <br />
<br />
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>"cis" proline</scene><br />
<scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene><br />
<br />
<br />
==='''Disulfide Bonds'''===<br />
Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/1'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/1'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/4'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/1'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 create an interaction between an alpha-helix and a beta sheet. This connection is the main contributor to the thermodynamic stability. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/3'>[C40,95]A</scene>, shows RNase A, missing the disulfide bond that would normally occur here, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/3'>Cys40-Cys95</scene>. As you can see in the variant, there are only 3 disulfide bonds present, shown in blue.<br />
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=='''References'''==</div>Diana Trautmann