Sandbox Reserved 199: Difference between revisions

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Using 2-dimensional 1H NMR, Udgaonkar et al. studied the folding pathway of bovine pancreatic Ribonuclease using an [http://en.wikipedia.org/wiki/Hydrogen-deuterium_exchange exchange reaction] between <scene name='Sandbox_Reserved_199/2aas_-_backbone_nitrogens/2'>deuterated peptide backbone amide protons</scene>  with solvent protons.  2- dimensional 1H NMR allowed for monitoring of proton exchange in the amide backbone for ten second time intervals, and this proton labeling could be terminated via a rapid drop in pH reaction conditions.  This research focused on the initial protein folding steps.
Using 2-dimensional 1H NMR, Udgaonkar et al. studied the folding pathway of bovine pancreatic Ribonuclease using an [http://en.wikipedia.org/wiki/Hydrogen-deuterium_exchange exchange reaction] between <scene name='Sandbox_Reserved_199/2aas_-_backbone_nitrogens/2'>deuterated peptide backbone amide protons</scene>  with solvent protons.  2- dimensional 1H NMR allowed for monitoring of proton exchange in the amide backbone for ten second time intervals, and this proton labeling could be terminated via a rapid drop in pH reaction conditions.  This research focused on the initial protein folding steps.


Starting with denatured wild-type RNase, it was hypothesized that as the peptide began to fold, the backbone amide proteins would become less energetically favorable to exchange protons with the solvent as the backbone amide protons became involved in folding-related intermolecular interactions (such as <scene name='Sandbox_Reserved_199/2aas_-_backbone_hydrogen_bondi/1'>hydrogen bonding</scene> ).
Starting with denatured wild-type RNaseA, it was hypothesized that as the peptide began to fold, the backbone amide proteins would become less energetically favorable to exchange protons with the solvent as the backbone amide protons became involved in folding-related intermolecular interactions (such as <scene name='Sandbox_Reserved_199/2aas_-_backbone_hydrogen_bondi/1'>hydrogen bonding</scene> ).


===Data and Results===
===Data and Results===
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===Experimental Procedure===
===Experimental Procedure===


This study builds on the results of the previously mentioned experiment. Using 3D NMR, Rico et al. obtained the first NMR 3-dimensional structures of Human Rnase 1. Using 20 different RNase NMR structures, researchers compared the RMSD values of key RNase 1 residues.  A residue with a high RMSD value has more flexibility and a residue’s ability to adapt to numerous conformations may be crucial to its active role within the enzyme.
Using 3D NMR, Rico et al. obtained the first NMR 3-dimensional structures of Human RNase 1. Using 20 different RNase, NMR structures, researchers compared the RMSD values of key RNase 1 residues.  A residue with a high RMSD value has more flexibility and a residue’s ability to adapt to numerous conformations may be crucial to its active role within the enzyme.
   
   
Furthermore, this study characterized the dimerizational prperties of wt Ribonuclease and mutated Ribonuclease variants.  Similar dimerization, observed in certain Bovine RNases, has shown to greatly enhance enzymatic activity and have augmented anti-tumoral action. Using varying enzyme concentrations and pH conditions as well as site-directed mutations, researchers were able to promote dimerization of RNase 1 variants.  
Furthermore, this study characterized the dimerization of wt Ribonuclease and mutated Ribonuclease variants.  Similar dimerization, observed in certain Bovine RNases, greatly enhances enzymatic activity and anti-tumoral action. Using varying enzyme concentrations, pH conditions and site-directed mutations, RNase 1 was dimerized.


This study also compares the location of certain residues in HcrI bound RNase 1 to unbound RNase. Through this comparison, these researchers provide greater insight into the catalytic mechanism and substrate specificity of RNase 1.
This study also compares the location of certain residues in bound RNase 1 to unbound RNase. This comparison, provides greater insight into the catalytic mechanism and substrate specificity of RNase 1.


[[Image:Kroupa RNase Dimer2.png|thumb |left |alt=X-Ray Diffraction RNase Dimer. |3F8G-Domain swapped dimer of human pancreatic Ribonuclease I.  Structure determined by X-Ray crystallography]]
[[Image:Kroupa RNase Dimer2.png|thumb |left |alt=X-Ray Diffraction RNase Dimer. |3F8G-Domain swapped dimer of human pancreatic Ribonuclease I.  Structure determined by X-Ray crystallography]]
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===Data and Results===
===Data and Results===


A 3D NMR structure was obtained with a backbone RMSD of 1.07Å. The obtained model shows a similar tertiary structure to the kidney bean shaped RNase A and is stabilized by four <scene name='Sandbox_Reserved_199/2k11_disulfide_bonds/1'>disulfide bonds</scene>. The structure shows three <scene name='Sandbox_Reserved_199/2k11_alpha_helix/2'>α-helices</scene> and seven <scene name='Sandbox_Reserved_199/2k11_beta_sheets/1'>β-sheets</scene>.  While this structure matches up fairly well with previous X-Ray crystallography structures of RNase 1, important differences in residue positioning can be seen in the <scene name='Sandbox_Reserved_199/2k11_all_models/3'>3D NMR structure</scene> which are not apparent in X-Ray crystallography. Specifically, certain residues with more flexibility undergo a significant conformational change when bound to certain substrates, such as the human ribonuclease inhibitor (HcrI).  These residues include: <scene name='Sandbox_Reserved_199/2k11_flexible_residues/1'>Arg 4, Lys 6, Arg 32, Arg 39, and Lys 102</scene>.  
A 3D NMR structure was obtained with a backbone RMSD of 1.07Å. The obtained model shows a similar tertiary structure to the kidney bean shaped RNase A and is stabilized by four <scene name='Sandbox_Reserved_199/2k11_disulfide_bonds/1'>disulfide bonds</scene>. The structure shows three <scene name='Sandbox_Reserved_199/2k11_alpha_helix/2'>α-helices</scene> and seven <scene name='Sandbox_Reserved_199/2k11_beta_sheets/1'>β-sheets</scene>.  While this structure matches up fairly well with previous X-Ray crystallography structures of RNase 1, important differences in residue positioning can be seen in the <scene name='Sandbox_Reserved_199/2k11_all_models/3'>3D NMR structure</scene> which are not apparent in X-Ray crystallography. Specifically, certain residues with more flexibility undergo a significant conformational change when bound to certain substrates, such as the human ribonuclease inhibitor (hRI).  These residues include: <scene name='Sandbox_Reserved_199/2k11_flexible_residues/1'>Arg 4, Lys 6, Arg 32, Arg 39, and Lys 102</scene>.  


This data suggests an “induced-fit” model of substrate binding and may prove vital to fully understanding RNase 1’s binding specificity for Hcrl; although two residues, <scene name='Sandbox_Reserved_199/2k11_42_43/1'>Pro 42 and Val43</scene>, show much more rigidity and possibly contribute some “lock-and-key” binding interaction.
This large conformational change suggests an “induced-fit” model of substrate binding and may prove vital to fully understanding RNase 1’s binding specificity for Hcrl; although two residues, <scene name='Sandbox_Reserved_199/2k11_42_43/1'>Pro 42 and Val43</scene>, show much more rigidity and possibly contribute some “lock-and-key” binding interaction. Designing cytotoxic variants of RNase1 may prove difficult due to the large number of active amino acids; numerous mutations may be required to fully "kill" the enyzme.  


[[Image:Kroupa Ribonuclease inhibitor.png|thumb |right |alt=RNase Inhibitor. |Example Ribonuclease inhibitor structure<ref>Willow. Top view of ribbon diagram of ribonuclease inhibitor (PDB accession code 2BNH). Made with MOLMOL. 2006. Web. 1 Apr. 2011..</ref>.]]
[[Image:Kroupa Ribonuclease inhibitor.png|thumb |right |alt=RNase Inhibitor. |Example Ribonuclease inhibitor structure<ref>Willow. Top view of ribbon diagram of ribonuclease inhibitor (PDB accession code 2BNH). Made with MOLMOL. 2006. Web. 1 Apr. 2011..</ref>.]]


15N NMR relaxation shows increased T1 values for the residues found in these sheets and loops (0.63-0.64s relative to 0.60s in helices). This suggests greater flexibility in these regions as well.  
15N NMR relaxation shows increased T1 values for the residues found in these sheets and loops (0.63-0.64s relative to 0.60s in helices), suggesting greater flexibility in these regions as well.  


Interestingly, the global correlation time of RNase 1 was shown to be much longer than what is expected for a 13.7 kDa monomer (10ns compared to 6-7ns). This shows that under NMR sample conditions, the RNase1 undergoes dimerization forming a monomer/dimer equilibrium.  Because dimerization has shown to have a significant effect on enzyme activity in bovine RNases, certain mutated RNase 1’s which show increased dimerization may be potential anti-cancer therapeutics.
Interestingly, the global correlation time of RNase 1 was much longer than expected for a 13.7 kDa monomer (10ns compared to 6-7ns) indicating the RNase1 undergoes dimerization forming a monomer/dimer equilibrium.  Because dimerization affects the enzymatic activity of bovine ribonuclease, RNase 1 variants with increased dimerization may be potential anti-cancer therapeutics.


== References ==
== References ==
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