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https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Mary+Liggett Proteopedia - User contributions [en] 2026-09-22T09:26:51Z User contributions MediaWiki 1.43.8 https://proteopedia.org/index.php?title=Sandbox_Reserved_1053&diff=2743053 Sandbox Reserved 1053 2017-04-21T17:10:03Z

<p>Mary Liggett: </p> <hr /> <div>==CzrA: A Zinc Dependent Transcriptional Regulator==<br /> <StructureSection load='2KJB' size='340' frame='true' side='right' caption='The dimer CzrA' scene=''><br /> == Background ==<br /> ===Operon Overview===<br /> [https://en.wikipedia.org/wiki/Operon Operons] are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of [https://en.wikipedia.org/wiki/RNA_polymerase RNA polymerase] to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. <br /> [[Image:Operon.png|500px|thumb|center|Figure 1:Overview of Operon Structure]]<br /> ===Czr Operon===<br /> The Chromosome Determined Zinc Responsible (Czr) operon acts as described above, with Czr A acting as the regulator protein. As such, Czr A is responsible for controlling the transcription of the rest of the operon and by extension, the transport of Zn <sup>2+</sup> out of the cell; the role of Czr A in the Czr operon is described in further detail as part of the explanation of biological function. In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon<ref name="critical">Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.</ref>. The Czr operon contains genes for the proteins Czr A and [http://proteopedia.org/wiki/index.php/3byr Czr B]. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup> (PDB code: 2kjb). In this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are aligned</scene>. Binding of zinc drives a conformational change (PDB code: 2kjc) in which the <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices become unaligned</scene>, lowering the affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA<ref name="critical"/>. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A<ref name="critical"/>, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell. <br /> <br /> == Structural Overview ==<br /> CzrA functions as a [https://en.wikipedia.org/wiki/Dimer_(chemistry) dimer]. The <scene name='69/694218/Monomeric_unit/1'>monomeric units</scene> dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>two beta sheets</scene> displayed in yellow. While the function of the [https://en.wikipedia.org/wiki/Beta_sheet beta sheets] are not yet known, key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA and Zn<sup> +2 </sup>. The <scene name='69/694220/2kjb_colored_alpha_4/1'>alpha 4 helices</scene> are the location of DNA binding and the <scene name='69/694220/Zinc_pocket_with_residues/1'>alpha 5 helics</scene> contain the Zn<sup> +2 </sup> binding site. As Zn<sup> +2 </sup> binds, the alpha 4 helices are <scene name='69/694220/2kjc_colored_alpha_4/1'>pushed out of alignment</scene>, repressing their DNA binding ability (Figure 2). Two seperate PDB codes exist for CzrA: CzrA with DNA bound (2KJB) and CzrA with zinc<sup>+2</sup> bound (2KJC). Unfortunately, zinc ions are not visible in the 2KJC NMR structure that was obtained for CzrA. <br /> [[Image:2KJB + 2KJC side by side.png|600px|center|thumb| Figure 2:Comparison of CzrA with Zn<sup>+2</sup> bound and CzrA with DNA bound with the alpha five helices shown in red and the alpha four helices shown in green]]<br /> <br /> == Binding of DNA ==<br /> The <scene name='69/694219/Serandhisresidues/3'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58 residues. <scene name='69/694220/2kjb_dna_alpha_4_helix/1'>These residues</scene> are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the alpha 4 helix (figure 3). The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by mutating the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref name="critical"/>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. <scene name='69/694220/Dna_binding_experiment/1'>Critical DNA binding residues</scene>, Gln53, Val42 (both shown in red), Ser54, Ser57, and His58 (all shown in orange), were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the <scene name='69/694220/Dna_residues_when_inhibited/1'>fully inhibited Zn<sup>2+</sup> bound state</scene>. The conformational change that occurs from the Zinc to DNA bound state regarding these residues is small, but the alpha 4 helix (shown in green in Figure 2) does subtly move. Because no major physical change occurs between these two states, it further supports that this region is the main DNA interaction site because of the loss of affinity after the mutation took place. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change.<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA.<br /> <br /> [[Image:DNABound Final.PNG|750px|thumb|center| Figure 3:Side by side rotation showing the main DNA groove in CzrA]]<br /> <br /> <br /> == Zinc Binding ==<br /> Many zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. CzrA fits into this category as an [https://en.wikipedia.org/wiki/Allosteric_regulation allosteric inhibitor] of the czr operon. Two [https://en.wikipedia.org/wiki/Zinc Zn<sup> +2</sup>] ions may bind to the dimer<ref name="critical"/>, at the location of the <scene name='69/694218/Alpha_5_helices/2'> alpha 5 </scene> helix from each monomer. As zinc binds, the alpha 5 helices <scene name='69/694218/2kjc_zinc_bound/1'>unalign</scene> to inhibit the DNA binding residues (Figure 2). Furthermore, CzrA must be in its dimer form for zinc to bind. The <scene name='69/694218/Spacefill_with_zinc_pockets/1'>zinc binding pocket</scene> is formed by two residues from each monomer, so Zn<sup>+2</sup> cannot bind to the monomer. The <scene name='69/694220/Zinc_binding_residues/6'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Zinc ions were not present in the solution NMR crystal structure, so a representation of a zinc ion in the binding pocket can be seen in figure 4. Histidines are a repetitive and commonly found residue in zinc-binding proteins <ref>Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun 4;4(6):1609-1614.</ref>.<br /> <br /> [[Image:Zinc tetrahedral complex.PNG|350px|thumb|center| Figure 4:Zn<sup>+2</sup> tetrahedral binding complex]]<br /> <br /> Zinc<sup>+2</sup> binding is driven by a large [https://en.wikipedia.org/wiki/Entropy entropic] gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 3). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell. <br /> <br /> </StructureSection><br /> <br /> __NOTOC__<br /> <br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1053&diff=2743044 Sandbox Reserved 1053 2017-04-21T17:05:34Z

<p>Mary Liggett: </p> <hr /> <div>==CzrA: A Zinc Dependent Transcriptional Regulator==<br /> <StructureSection load='2KJB' size='340' frame='true' side='right' caption='The dimer CzrA' scene=''><br /> == Background ==<br /> ===Operon Overview===<br /> [https://en.wikipedia.org/wiki/Operon Operons] are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of [https://en.wikipedia.org/wiki/RNA_polymerase RNA polymerase] to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. <br /> [[Image:Operon.png|500px|thumb|center|Figure 1:Overview of Operon Structure]]<br /> ===Czr Operon===<br /> The Chromosome Determined Zinc Responsible (Czr) operon acts as described above, with Czr A acting as the regulator protein. As such, Czr A is responsible for controlling the transcription of the rest of the operon and by extension, the transport of Zn <sup>2+</sup> out of the cell; the role of Czr A in the Czr operon is described in further detail as part of the explanation of biological function. In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon<ref name="critical">Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.</ref>. The Czr operon contains genes for the proteins Czr A and [http://proteopedia.org/wiki/index.php/3byr Czr B]. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup> (PDB code: 2kjb). In this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are aligned</scene>. Binding of zinc drives a conformational change (PDB code: 2kjc) in which the <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices become unaligned</scene>, lowering the affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA<ref name="critical"/>. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A<ref name="critical"/>, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell. <br /> <br /> == Structural Overview ==<br /> CzrA functions as a [https://en.wikipedia.org/wiki/Dimer_(chemistry) dimer]. The <scene name='69/694218/Monomeric_unit/1'>monomeric units</scene> dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>two beta sheets</scene> displayed in yellow. While the function of the [https://en.wikipedia.org/wiki/Beta_sheet beta sheets] are not yet known, key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA and Zn<sup> +2 </sup>. The <scene name='69/694220/2kjb_colored_alpha_4/1'>alpha 4 helices</scene> are the location of DNA binding and the <scene name='69/694220/Zinc_pocket_with_residues/1'>alpha 5 helics</scene> contain the Zn<sup> +2 </sup> binding site. As Zn<sup> +2 </sup> binds, the alpha 4 helices are <scene name='69/694220/2kjc_colored_alpha_4/1'>pushed out of alignment</scene>, repressing their DNA binding ability (Figure 2). Two seperate PDB codes exist for CzrA: CzrA with DNA bound (2KJB) and CzrA with zinc<sup>+2</sup> bound (2KJC). Unfortunately, zinc ions are not visible in the 2KJC NMR structure that was obtained for CzrA. <br /> [[Image:2KJB + 2KJC side by side.png|600px|center|thumb| Figure 2:Comparison of CzrA with Zn<sup>+2</sup> bound and CzrA with DNA bound with the alpha five helices shown in red and the alpha four helices shown in green]]<br /> <br /> == Binding of DNA ==<br /> The <scene name='69/694219/Serandhisresidues/3'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58 residues. <scene name='69/694220/2kjb_dna_alpha_4_helix/1'>These residues</scene>are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the alpha 4 helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by mutating the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref name="critical"/>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. <scene name='69/694220/Dna_binding_experiment/1'>Critical DNA binding residues</scene>, Gln53, Val42 (both shown in red), Ser54, Ser57, and His58 (all shown in orange), were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the <scene name='69/694220/Dna_residues_when_inhibited/1'>fully inhibited Zn<sup>2+</sup> bound state</scene>. The conformational change that occurs from the Zinc to DNA bound state regarding these residues is small, but the alpha 4 helix (shown in green in Figure 2) does subtly move. Because no major physical change occurs between these two states, it further supports that this region is the main DNA interaction site because of the loss of affinity after the mutation took place. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change.<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA.<br /> <br /> [[Image:DNABound Final.PNG|850px|thumb|center| Figure 3:Side by side rotation showing the main DNA groove in CzrA]]<br /> <br /> <br /> == Zinc Binding ==<br /> Many zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. CzrA fits into this category as an [https://en.wikipedia.org/wiki/Allosteric_regulation allosteric inhibitor] of the czr operon. Two [https://en.wikipedia.org/wiki/Zinc Zn<sup> +2</sup>] ions may bind to the dimer<ref name="critical"/>, at the location of the <scene name='69/694218/Alpha_5_helices/2'> alpha 5 </scene> helix from each monomer. As zinc binds, the alpha 5 helices <scene name='69/694218/2kjc_zinc_bound/1'>unalign</scene> to inhibit the DNA binding residues (Figure 2). Furthermore, CzrA must be in its dimer form for zinc to bind. The <scene name='69/694218/Spacefill_with_zinc_pockets/1'>zinc binding pocket</scene> is formed by two residues from each monomer, so Zn<sup>+2</sup> cannot bind to the monomer. The <scene name='69/694220/Zinc_binding_residues/6'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Zinc ions were not present in the solution NMR crystal structure, so a representation of a zinc ion in the binding pocket can be seen in figure 3. Histidines are a repetitive and commonly found residue in zinc-binding proteins <ref>Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun 4;4(6):1609-1614.</ref>.<br /> <br /> [[Image:Zinc tetrahedral complex.PNG|350px|thumb|center| Figure 4:Zn<sup>+2</sup> tetrahedral binding complex]]<br /> <br /> Zinc<sup>+2</sup> binding is driven by a large [https://en.wikipedia.org/wiki/Entropy entropic] gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 3). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell. <br /> <br /> </StructureSection><br /> <br /> __NOTOC__<br /> <br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=File:DNABound_Final.PNG&diff=2743040 File:DNABound Final.PNG 2017-04-21T17:00:00Z

<p>Mary Liggett: </p> <hr /> <div></div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1053&diff=2740471 Sandbox Reserved 1053 2017-04-18T12:56:07Z

<p>Mary Liggett: </p> <hr /> <div>==CzrA: A Zinc Dependent Transcriptional Regulator==<br /> <StructureSection load='2KJB' size='340' frame='true' side='right' caption='The dimer CzrA' scene=''><br /> == Background ==<br /> ===Operon Overview===<br /> [https://en.wikipedia.org/wiki/Operon Operons] are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of [https://en.wikipedia.org/wiki/RNA_polymerase RNA polymerase] to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. <br /> [[Image:Operon.png|500px|thumb|center|Figure 1:Overview of Operon Structure]]<br /> ===Czr Operon===<br /> The Chromosome Determined Zinc Responsible (Czr) operon acts as described above, with Czr A acting as the regulator protein. As such, Czr A is responsible for controlling the transcription of the rest of the operon and by extension, the transport of Zn <sup>2+</sup> out of the cell; the role of Czr A in the Czr operon is described in further detail as part of the explanation of biological function. In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon<ref name="critical">Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.</ref>. The Czr operon contains genes for the proteins Czr A and [http://proteopedia.org/wiki/index.php/3byr Czr B]. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>. In this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are aligned</scene>. Binding of zinc drives a conformational change in which the <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices become unaligned</scene>, lowering the affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA<ref name="critical"/>. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A<ref name="critical"/>, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell. <br /> <br /> == Structural Overview ==<br /> CzrA functions as a [https://en.wikipedia.org/wiki/Dimer_(chemistry) dimer]. The <scene name='69/694218/Monomeric_unit/1'>monomeric units</scene> dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>two beta sheets</scene> displayed in yellow. While the function of the [https://en.wikipedia.org/wiki/Beta_sheet beta sheets] are not yet known, key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA and Zn<sup> +2 </sup>. The <scene name='69/694220/2kjb_colored_alpha_4/1'>alpha 4 helices</scene> are the location of DNA binding and the <scene name='69/694220/Zinc_pocket_with_residues/1'>alpha 5 helics</scene> contain the Zn<sup> +2 </sup> binding site. As Zn<sup> +2 </sup> binds, the alpha 4 helices are <scene name='69/694220/2kjc_colored_alpha_4/1'>pushed out of alignment</scene>, repressing their DNA binding ability (Figure 2). Unfortunately, zinc ions are not visible in the NMR structure that was obtained for CzrA.<br /> [[Image:2KJB + 2KJC side by side.png|600px|center|thumb| Figure 2:Comparison of CzrA with Zn<sup>+2</sup> bound and CzrA with DNA bound with the alpha five helices shown in red and the alpha four helices shown in green]]<br /> <br /> == Binding of DNA ==<br /> The <scene name='69/694219/Serandhisresidues/3'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58 residues. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the alpha 4 helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by mutating the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref name="critical"/>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. <scene name='69/694220/Dna_binding_experiment/1'>Critical DNA binding residues</scene>, Gln53, Val42 (both shown in red), Ser54, Ser57, and His58 (all shown in orange), were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the <scene name='69/694220/Dna_residues_when_inhibited/1'>fully inhibited Zn<sup>2+</sup> bound state</scene>. The conformational change that occurs from the Zinc to DNA bound state regarding these residues is small, but the alpha 4 helix (shown in green in Figure 2) does subtly move. Because no major physical change occurs between these two states, it further supports that this region is the main DNA interaction site because of the loss of affinity after the mutation took place. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change.<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA.<br /> <br /> <br /> == Zinc Binding ==<br /> Many zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. CzrA fits into this category as an [https://en.wikipedia.org/wiki/Allosteric_regulation allosteric inhibitor] of the czr operon. Two [https://en.wikipedia.org/wiki/Zinc Zn<sup> +2</sup>] ions may bind to the dimer<ref name="critical"/>, at the location of the <scene name='69/694218/Alpha_5_helices/2'> alpha 5 </scene> helix from each monomer. As zinc binds, the alpha 5 helices <scene name='69/694218/2kjc_zinc_bound/1'>unalign</scene> to inhibit the DNA binding residues (Figure 2). Furthermore, CzrA must be in its dimer form for zinc to bind. The <scene name='69/694218/Spacefill_with_zinc_pockets/1'>zinc binding pocket</scene> is formed by two residues from each monomer, so Zn<sup>+2</sup> cannot bind to the monomer. The <scene name='69/694220/Zinc_binding_residues/6'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Zinc ions were not present in the solution NMR crystal structure, so a representation of a zinc ion in the binding pocket can be seen in figure 3. Histidines are a repetitive and commonly found residue in zinc-binding proteins <ref>Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun 4;4(6):1609-1614.</ref>.<br /> <br /> Zinc<sup>+2</sup> binding is driven by a large [https://en.wikipedia.org/wiki/Entropy entropic] gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 3). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell. <br /> [[Image:Zinc tetrahedral complex.PNG|350px|thumb|center| Figure 3:Zn<sup>+2</sup> tetrahedral binding complex]]<br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1053&diff=2740454 Sandbox Reserved 1053 2017-04-18T12:41:25Z

<p>Mary Liggett: </p> <hr /> <div>{{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<br /> ==CzrA: A Zinc Dependent Transcriptional Regulator==<br /> <StructureSection load='2KJB' size='340' side='right' caption='The dimer CzrA' scene=''><br /> <br /> == Background ==<br /> ===Operon Overview===<br /> [https://en.wikipedia.org/wiki/Operon Operons] are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of [https://en.wikipedia.org/wiki/RNA_polymerase RNA polymerase] to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. <br /> [[Image:Operon.png|500px|thumb|center|Figure 1:Overview of Operon Structure]]<br /> ===Czr Operon===<br /> The Chromosome Determined Zinc Responsible (Czr) operon acts as described above, with Czr A acting as the regulator protein. As such, Czr A is responsible for controlling the transcription of the rest of the operon and by extension, the transport of Zn <sup>2+</sup> out of the cell; the role of Czr A in the Czr operon is described in further detail as part of the explanation of biological function. In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> <br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon<ref name="critical">Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.</ref>. The Czr operon contains genes for the proteins Czr A and [http://proteopedia.org/wiki/index.php/3byr Czr B]. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>. In this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are aligned</scene>. Binding of zinc drives a conformational change in which the <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices become unaligned</scene>, lowering the affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA<ref name="critical"/>. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A<ref name="critical"/>, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell. <br /> <br /> == Structural Overview ==<br /> CzrA functions as a [https://en.wikipedia.org/wiki/Dimer_(chemistry) dimer]. The <scene name='69/694218/Monomeric_unit/1'>monomeric units</scene> dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>two beta sheets</scene> displayed in yellow. While the function of the [https://en.wikipedia.org/wiki/Beta_sheet beta sheets] are not yet known, key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA and Zn<sup> +2 </sup>. The <scene name='69/694220/2kjb_colored_alpha_4/1'>alpha 4 helices</scene> are the location of DNA binding and the <scene name='69/694220/Zinc_pocket_with_residues/1'>alpha 5 helics</scene> contain the Zn<sup> +2 </sup> binding site. As Zn<sup> +2 </sup> binds, the alpha 4 helices are <scene name='69/694220/2kjc_colored_alpha_4/1'>pushed out of alignment</scene>, repressing their DNA binding ability (Figure 2). Unfortunately, zinc ions are not visible in the NMR structure that was obtained for CzrA.<br /> [[Image:2KJB + 2KJC side by side.png|600px|center|thumb| Figure 2:Comparison of CzrA with Zn<sup>+2</sup> bound and CzrA with DNA bound with the alpha five helices shown in red and the alpha four helices shown in green]]<br /> <br /> == Binding of DNA ==<br /> The <scene name='69/694219/Serandhisresidues/3'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58 residues. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the alpha 4 helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by mutating the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref name="critical"/>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. <scene name='69/694220/Dna_binding_experiment/1'>Critical DNA binding residues</scene>, Gln53, Val42 (both shown in red), Ser54, Ser57, and His58 (all shown in orange), were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. While no major physical change occurred when these mutations were made, the significance can be seen in the loss on binding affinity. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change.<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA.<br /> <br /> <br /> == Zinc Binding ==<br /> Many zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. CzrA fits into this category as an [https://en.wikipedia.org/wiki/Allosteric_regulation allosteric inhibitor] of the czr operon. Two [https://en.wikipedia.org/wiki/Zinc Zn<sup> +2</sup>] ions may bind to the dimer<ref name="critical"/>, at the location of the <scene name='69/694218/Alpha_5_helices/2'> alpha 5 </scene> helix from each monomer. As zinc binds, the alpha 5 helices <scene name='69/694218/2kjc_zinc_bound/1'>unalign</scene> to inhibit the DNA binding residues (Figure 2). Furthermore, CzrA must be in its dimer form for zinc to bind. The <scene name='69/694218/Spacefill_with_zinc_pockets/1'>zinc binding pocket</scene> is formed by two residues from each monomer, so Zn<sup>+2</sup> cannot bind to the monomer. The <scene name='69/694220/Zinc_binding_residues/6'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Zinc ions were not present in the solution NMR crystal structure, so a representation of a zinc ion in the binding pocket can be seen in figure 3. Histidines are a repetitive and commonly found residue in zinc-binding proteins <ref>Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun 4;4(6):1609-1614.</ref>.<br /> <br /> Zinc<sup>+2</sup> binding is driven by a large [https://en.wikipedia.org/wiki/Entropy entropic] gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 3). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell. <br /> [[Image:Zinc tetrahedral complex.PNG|350px|thumb|center| Figure 3:Zn<sup>+2</sup> tetrahedral binding complex]]<br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1053&diff=2740441 Sandbox Reserved 1053 2017-04-18T12:28:44Z

<p>Mary Liggett: </p> <hr /> <div>{{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<br /> ==CzrA: A Zinc Dependent Transcriptional Regulator==<br /> <StructureSection load='2KJB' size='340' side='right' caption='The dimer CzrA' scene=''><br /> <br /> == Background ==<br /> ===Operon Overview===<br /> [https://en.wikipedia.org/wiki/Operon Operons] are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of [https://en.wikipedia.org/wiki/RNA_polymerase RNA polymerase] to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. <br /> [[Image:Operon.png|500px|thumb|center|Figure 1:Overview of Operon Structure]]<br /> ===Czr Operon===<br /> The Chromosome Determined Zinc Responsible (Czr) operon acts as described above, with Czr A acting as the regulator protein. As such, Czr A is responsible for controlling the transcription of the rest of the operon and by extension, the transport of Zn <sup>2+</sup> out of the cell; the role of Czr A in the Czr operon is described in further detail as part of the explanation of biological function. In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> <br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon<ref name="critical">Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.</ref>. The Czr operon contains genes for the proteins Czr A and [http://proteopedia.org/wiki/index.php/3byr Czr B]. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>. In this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are aligned</scene>. Binding of zinc drives a conformational change in which the <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices become unaligned</scene>, lowering the affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA<ref name="critical"/>. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A<ref name="critical"/>, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell. <br /> <br /> == Structural Overview ==<br /> CzrA functions as a [https://en.wikipedia.org/wiki/Dimer_(chemistry) dimer]. The <scene name='69/694218/Monomeric_unit/1'>monomeric units</scene> dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>two beta sheets</scene> displayed in yellow. While the function of the [https://en.wikipedia.org/wiki/Beta_sheet beta sheets] are not yet known, key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA and Zn<sup> +2 </sup>. The <scene name='69/694220/2kjb_colored_alpha_4/1'>alpha 4 helices</scene> are the location of DNA binding and the <scene name='69/694220/Zinc_pocket_with_residues/1'>alpha 5 helics</scene> contain the Zn<sup> +2 </sup> binding site. As Zn<sup> +2 </sup> binds, the alpha 4 helices are <scene name='69/694220/2kjc_colored_alpha_4/1'>pushed out of alignment</scene>, repressing their DNA binding ability (Figure 2). Unfortunately, zinc ions are not visible in the NMR structure that was obtained for CzrA.<br /> [[Image:2KJB + 2KJC side by side.png|600px|center|thumb| Figure 2:Comparison of CzrA with Zn<sup>+2</sup> bound and CzrA with DNA bound with the alpha five helices shown in red and the alpha four helices shown in green]]<br /> <br /> == Binding of DNA ==<br /> The <scene name='69/694219/Serandhisresidues/3'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58 residues. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the alpha 4 helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by mutating the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref name="critical"/>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. <scene name='69/694220/Dna_binding_experiment/1'>Critical DNA binding residues</scene>, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change.<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA.<br /> <br /> <br /> == Zinc Binding ==<br /> Many zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. CzrA fits into this category as an [https://en.wikipedia.org/wiki/Allosteric_regulation allosteric inhibitor] of the czr operon. Two [https://en.wikipedia.org/wiki/Zinc Zn<sup> +2</sup>] ions may bind to the dimer<ref name="critical"/>, at the location of the <scene name='69/694218/Alpha_5_helices/2'> alpha 5 </scene> helix from each monomer. As zinc binds, the alpha 5 helices <scene name='69/694218/2kjc_zinc_bound/1'>unalign</scene> to inhibit the DNA binding residues. Furthermore, CzrA must be in its dimer form for zinc to bind. The <scene name='69/694218/Spacefill_with_zinc_pockets/1'>zinc binding pocket</scene> is formed by two residues from each monomer, so Zn<sup>+2</sup> cannot bind to the monomer. The <scene name='69/694220/Zinc_binding_residues/5'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Zinc ions were not present in the solution NMR crystal structure, so a representation of a zinc ion in the binding pocket can be seen in figure 3. Histidines are a repetitive and commonly found residue in zinc-binding proteins <ref>Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun 4;4(6):1609-1614.</ref>.<br /> <br /> Zinc<sup>+2</sup> binding is driven by a large [https://en.wikipedia.org/wiki/Entropy entropic] gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 3). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell. <br /> [[Image:Zinc tetrahedral complex.PNG|350px|thumb|center| Figure 3:Zn<sup>+2</sup> tetrahedral binding complex]]<br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2738266 Sandbox Reserved 1052 2017-04-11T12:13:47Z

<p>Mary Liggett: </p> <hr /> <div>'''Bold text'''<Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> == Background ==<br /> [https://en.wikipedia.org/wiki/Operon Operons] are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of [https://en.wikipedia.org/wiki/RNA_polymerase RNA polymerase] to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. The Chromosome Determined Zinc Responsible (Czr) operon acts in exactly this manner, Czr A specifically is the regulator protein. The role of Czr A in the Czr operon is described in further detail under biological function.<br /> In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> [[Image:Operon.png|600px|thumb|center|Visual of Overview of Operon Structure]]<br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon<ref name="critical">Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.</ref>. The Czr operon contains genes for the proteins Czr A and [http://proteopedia.org/wiki/index.php/3byr Czr B]. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>, in this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are open</scene>. The <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices swing down</scene> to achieve the other conformation which binds two Zn<sup>2+</sup> ions and has relatively low affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA<ref name="critical"/>. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A<ref name="critical"/>, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell. <br /> <br /> == Structural Overview ==<br /> CzrA functions as a [https://en.wikipedia.org/wiki/Dimer_(chemistry) dimer]. The <scene name='69/694218/Monomeric_unit/1'>monomeric units</scene> dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>two beta sheets</scene> displayed in yellow. While the function of the [https://en.wikipedia.org/wiki/Beta_sheet beta sheets] are not yet known, key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA and Zn<sup> +2 </sup>. The <scene name='69/694218/Alpha_4_helix/1'>alpha 4 helix</scene> is the location of DNA binding and the <scene name='69/694218/Alpha_5_helix/1'>alpha 5 helix</scene> contains the Zn<sup> +2 </sup> binding site. As Zn<sup> +2 </sup> binds, the alpha 4 helices are <scene name='69/694218/Alpha_4_helices_pushed/1'>pushed out of alignment</scene>, repressing their DNA binding ability.<br /> <br /> == Binding of DNA ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58 residues. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by mutating the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref name="critical"/>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA.<br /> <br /> <br /> == Zinc Binding ==<br /> Most zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. CzrA fits into this category as an [https://en.wikipedia.org/wiki/Allosteric_regulation allosteric inhibitor] of the czr operon. Two [https://en.wikipedia.org/wiki/Zinc Zn<sup> +2</sup>] ions may bind to the dimer<ref name="critical"/>, at the location of the <scene name='69/694218/Alpha_5_helices/2'> alpha 5 </scene> helix from each monomer. As zinc binds, the alpha 5 helices <scene name='69/694218/2kjc_zinc_bound/1'>swing down</scene> to inhibit the DNA binding residues. Furthermore, CzrA must be in its dimer form for zinc to bind. The <scene name='69/694218/Spacefill_with_zinc_pockets/1'>zinc binding pocket</scene> is formed by two residues from each monomer, so Zn<sup>+2</sup> cannot bind to the monomer. The <scene name='69/694218/Zinc_residues/1'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Histidines are a repetitive and commonly found residue in zinc-binding proteins <ref>Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun 4;4(6):1609-1614.</ref>.<br /> <br /> Zinc<sup>+2</sup> binding is driven by a large [https://en.wikipedia.org/wiki/Entropy entropic] gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 1). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell. <br /> [[Image:Zinc tetrahedral complex.PNG|thumb|center| Figure 1:Zn<sup>+2</sup> tetrahedral binding complex]]<br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1053&diff=2734588 Sandbox Reserved 1053 2017-03-31T19:06:46Z

<p>Mary Liggett: </p> <hr /> <div>{{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<br /> ==CzrA==<br /> <StructureSection load='2KJB' size='340' side='right' caption='The dimer CzrA' scene=''><br /> <br /> == Background ==<br /> [https://en.wikipedia.org/wiki/Operon Operons] are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of [https://en.wikipedia.org/wiki/RNA_polymerase RNA polymerase] to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. The Chromosome Determined Zinc Responsible (Czr) operon acts in exactly this manner, Czr A specifically is the regulator protein. The role of Czr A in the Czr operon is described in further detail under biological function.<br /> In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> [[Image:Operon.png|600px|thumb|center|Visual of Overview of Operon Structure]]<br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon<ref name="critical">Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.</ref>. The Czr operon contains genes for the proteins Czr A and [http://proteopedia.org/wiki/index.php/3byr Czr B]. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>, in this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are open</scene>. The <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices swing down</scene> to achieve the other conformation which binds two Zn<sup>2+</sup> ions and has relatively low affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA<ref name="critical"/>. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A<ref name="critical"/>, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell. <br /> <br /> == Structural Overview ==<br /> CzrA functions as a [https://en.wikipedia.org/wiki/Dimer_(chemistry) dimer]. The <scene name='69/694218/Monomeric_unit/1'>monomeric units</scene> dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>two beta sheets</scene> displayed in yellow. While the function of the [https://en.wikipedia.org/wiki/Beta_sheet beta sheets] are not yet known, key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA and Zn<sup> +2 </sup>. The <scene name='69/694218/Alpha_4_helix/1'>alpha 4 helix</scene> is the location of DNA binding and the <scene name='69/694218/Alpha_5_helix/1'>alpha 5 helix</scene> contains the Zn<sup> +2 </sup> binding site. As Zn<sup> +2 </sup> binds, the alpha 4 helices are <scene name='69/694218/Alpha_4_helices_pushed/1'>pushed out of alignment</scene>, repressing their DNA binding ability.<br /> <br /> == Binding of DNA ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58 residues. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by mutating the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref name="critical"/>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA.<br /> <br /> <br /> == Zinc Binding ==<br /> Most zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. CzrA fits into this category as an [https://en.wikipedia.org/wiki/Allosteric_regulation allosteric inhibitor] of the czr operon. Two [https://en.wikipedia.org/wiki/Zinc Zn<sup> +2</sup>] ions may bind to the dimer<ref name="critical"/>, at the location of the <scene name='69/694218/Alpha_5_helices/2'> alpha 5 </scene> helix from each monomer. As zinc binds, the alpha 5 helices <scene name='69/694218/2kjc_zinc_bound/1'>swing down</scene> to inhibit the DNA binding residues. Furthermore, CzrA must be in its dimer form for zinc to bind. The <scene name='69/694218/Spacefill_with_zinc_pockets/1'>zinc binding pocket</scene> is formed by two residues from each monomer, so Zn<sup>+2</sup> cannot bind to the monomer. The <scene name='69/694218/Zinc_residues/1'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Histidines are a repetitive and commonly found residue in zinc-binding proteins <ref>Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun 4;4(6):1609-1614.</ref>.<br /> <br /> Zinc<sup>+2</sup> binding is driven by a large [https://en.wikipedia.org/wiki/Entropy entropic] gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 1). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell. <br /> [[Image:Zinc tetrahedral complex.PNG|thumb|center| Figure 1:Zn<sup>+2</sup> tetrahedral binding complex]]<br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1053&diff=2734585 Sandbox Reserved 1053 2017-03-31T18:59:53Z

<p>Mary Liggett: </p> <hr /> <div>{{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<br /> ==CzrA==<br /> <StructureSection load='2KJB' size='340' side='right' caption='The dimer CzrA' scene=''><br /> <br /> == Background ==<br /> [https://en.wikipedia.org/wiki/Operon Operons] are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of [https://en.wikipedia.org/wiki/RNA_polymerase RNA polymerase] to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. The Chromosome Determined Zinc Responsible (Czr) operon acts in exactly this manner, Czr A specifically is the regulator protein. The role of Czr A in the Czr operon is described in further detail under biological function.<br /> In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> [[Image:Operon.png|600px|thumb|center|Visual of Overview of Operon Structure]]<br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon<ref>DOI:10.1073pnas.0905558106</ref>. The Czr operon contains genes for the proteins Czr A and [http://proteopedia.org/wiki/index.php/3byr Czr B]. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>, in this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are open</scene>. The <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices swing down</scene> to achieve the other conformation which binds two Zn<sup>2+</sup> ions and has relatively low affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA<ref>DOI:10.1073pnas.0905558106</ref>. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A<ref>DOI:10.1073pnas.0905558106</ref>, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell. <br /> <br /> == Structural Overview ==<br /> CzrA functions as a [https://en.wikipedia.org/wiki/Dimer_(chemistry) dimer]. The <scene name='69/694218/Monomeric_unit/1'>monomeric units</scene> dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>two beta sheets</scene> displayed in yellow. While the function of the [https://en.wikipedia.org/wiki/Beta_sheet beta sheets] are not yet known, key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA and Zn<sup> +2 </sup>. The <scene name='69/694218/Alpha_4_helix/1'>alpha 4 helix</scene> is the location of DNA binding and the <scene name='69/694218/Alpha_5_helix/1'>alpha 5 helix</scene> contains the Zn<sup> +2 </sup> binding site. As Zn<sup> +2 </sup> binds, the alpha 4 helices are <scene name='69/694218/Alpha_4_helices_pushed/1'>pushed out of alignment</scene>, repressing their DNA binding ability.<br /> <br /> == Binding of DNA ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58 residues. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by mutating the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref>DOI:10.1073pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA.<br /> <br /> <br /> == Zinc Binding ==<br /> Most zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. CzrA fits into this category as an [https://en.wikipedia.org/wiki/Allosteric_regulation allosteric inhibitor] of the czr operon. Two [https://en.wikipedia.org/wiki/Zinc Zn<sup> +2</sup>] ions may bind to the dimer<ref>DOI:10.1073/pnas.0636943100</ref>, at the location of the <scene name='69/694218/Alpha_5_helices/2'> alpha 5 </scene> helix from each monomer. As zinc binds, the alpha 5 helices <scene name='69/694218/2kjc_zinc_bound/1'>swing down</scene> to inhibit the DNA binding residues. Furthermore, CzrA must be in its dimer form for zinc to bind. The <scene name='69/694218/Spacefill_with_zinc_pockets/1'>zinc binding pocket</scene> is formed by two residues from each monomer, so Zn<sup>+2</sup> cannot bind to the monomer. The <scene name='69/694218/Zinc_residues/1'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Histidines are a repetitive and commonly found residue in zinc-binding proteins <ref>Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun 4;4(6):1609-1614.</ref>.<br /> <br /> Zinc<sup>+2</sup> binding is driven by a large [https://en.wikipedia.org/wiki/Entropy entropic] gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 1). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell. <br /> [[Image:Zinc tetrahedral complex.PNG|thumb|center| Figure 1:Zn<sup>+2</sup> tetrahedral binding complex]]<br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732652 Sandbox Reserved 1052 2017-03-28T13:31:20Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> == Background ==<br /> Operons are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of RNA polymerase to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. The Chromosome Determined Zinc Responsible (Czr) operon acts in exactly this manner, Czr A specifically is the regulator protein. The role of Czr A in the Czr operon is described in further detail under biological function.<br /> In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon. The Czr operon contains genes for the proteins Czr A and Czr B. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>, in this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are open</scene>. The <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices swing down</scene> to achieve the other conformation which binds two Zn<sup>2+</sup> ions and has relatively low affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell.<br /> <br /> == Structural Overview ==<br /> CzrA functions as a dimer. The <scene name='69/694218/Monomeric_unit/1'>monomeric units</scene> dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>two beta sheets</scene> displayed in yellow. While the function of the beta sheets are not yet known, key helices regulate the binding of DNA and Zn<sup> +2 </sup>. The <scene name='69/694218/Alpha_4_helix/1'>alpha 4 helix</scene> is the location of DNA binding and the <scene name='69/694218/Alpha_5_helix/1'>alpha 5 helix</scene> contains the Zn<sup> +2 </sup> binding site. As Zn<sup> +2 </sup> binds, the alpha 4 helices are <scene name='69/694218/Alpha_4_helices_pushed/1'>pushed out of alignment</scene>, repressing their DNA binding ability.<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref>DOI: 10.1073pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Binding ==<br /> Most zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. CzrA fits into this category as an allosteric inhibitor of the czr operon. Two Zn<sup> +2 </sup> ions may bind to the dimer<ref>DOI:10.1073/pnas.0636943100</ref>, at the location of the <scene name='69/694218/Alpha_5_helices/2'> alpha 5 </scene> helix from each monomer. As zinc binds, the alpha 5 helices <scene name='69/694218/2kjc_zinc_bound/1'>swing down</scene> to inhibit the DNA binding residues. Furthermore, CzrA must be in its dimer form for zinc to bind. The <scene name='69/694218/Spacefill_with_zinc_pockets/1'>zinc binding pocket</scene> is formed by two residues from each monomer, so Zn<sup>+2</sup> cannot bind to the monomer. The <scene name='69/694218/Zinc_residues/1'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Histidines are a repetitive and commonly found residue in zinc-binding proteins <ref>Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun 4;4(6):1609-1614.</ref>.<br /> <br /> Zinc<sup>+2</sup> binding is driven by a large entropic gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 1). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell. <br /> [[Image:Zinc tetrahedral complex.PNG|thumb|center| Figure 1:Zn<sup>+2</sup> tetrahedral binding complex]]<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> 1. Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182 DOI: 10.1073pnas.0905558106<br /> <br /> 2. Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376 DOI: 10.1021/ja208047b</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732637 Sandbox Reserved 1052 2017-03-28T13:17:33Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> == Background ==<br /> Operons are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of RNA polymerase to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. The Chromosome Determined Zinc Responsible (Czr) operon acts in exactly this manner, Czr A specifically is the regulator protein. The role of Czr A in the Czr operon is described in further detail under biological function.<br /> In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon. The Czr operon contains genes for the proteins Czr A and Czr B. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>, in this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are open</scene>. The <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices swing down</scene> to achieve the other conformation which binds two Zn<sup>2+</sup> ions and has relatively low affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell.<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref>DOI: 10.1073pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> 1. Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182 DOI: 10.1073pnas.0905558106<br /> <br /> 2. Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376 DOI: 10.1021/ja208047b</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732634 Sandbox Reserved 1052 2017-03-28T13:15:42Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> == Background ==<br /> Operons are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of RNA polymerase to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. The Chromosome Determined Zinc Responsible (Czr) operon acts in exactly this manner, Czr A specifically is the regulator protein. The role of Czr A in the Czr operon is described in further detail under biological function.<br /> In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon. The Czr operon contains genes for the proteins Czr A and Czr B. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>, in this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are open</scene>. The <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices swing down</scene> to achieve the other conformation which binds two Zn<sup>2+</sup> ions and has relatively low affinity for DNA<ref>DOI: 10.1073pnas.0905558106</ref>. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell<ref>DOI: 10.1073pnas.0905558106</ref>.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell<ref>DOI: 10.1073pnas.0905558106</ref>.<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref>DOI: 10.1073pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> 1. Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182 DOI: 10.1073pnas.0905558106<br /> <br /> 2. Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376 DOI: 10.1021/ja208047b</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732629 Sandbox Reserved 1052 2017-03-28T13:09:12Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> == Background ==<br /> Operons are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of RNA polymerase to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. The Chromosome Determined Zinc Responsible (Czr) operon acts in exactly this manner, Czr A specifically is the regulator protein. The role of Czr A in the Czr operon is described in further detail under biological function.<br /> In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.<br /> <br /> == Biological Function ==<br /> Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon. The Czr operon contains genes for the proteins Czr A and Czr B. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>, in this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are open</scene>. The <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices swing down</scene> to achieve the other conformation which binds two Zn<sup>2+</sup> ions and has relatively low affinity for DNA. <br /> ===DNA Binding ===<br /> Czr A performs it's primary function when bound to DNA. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.<br /> ===Zinc Binding ===<br /> Zinc acts as an inhibitor to Czr A, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell.<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref>DOI: 10.1073pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> 1. Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182 DOI: 10.1073pnas.0905558106<br /> <br /> 2. Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376 DOI: 10.1021/ja208047b</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732612 Sandbox Reserved 1052 2017-03-28T12:55:43Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref>DOI: 10.1073pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> 1. Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182 DOI: 10.1073pnas.0905558106<br /> <br /> 2. Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376 DOI: 10.1021/ja208047b</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732604 Sandbox Reserved 1052 2017-03-28T12:51:27Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref>DOI: 10.1073pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> 1. Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182<br /> <br /> 2. Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732576 Sandbox Reserved 1052 2017-03-28T12:25:54Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref>DOI: 10.1073pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this article show the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> 1. Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182<br /> <br /> 2. Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732574 Sandbox Reserved 1052 2017-03-28T12:24:55Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref>DOI: 10.1073pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this article show the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182<br /> <br /> Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732566 Sandbox Reserved 1052 2017-03-28T12:17:02Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref>DOI: 10.1073pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this article show the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<ref>DOI: 10.1073pnas.0905558106</ref><br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182<br /> <br /> Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732537 Sandbox Reserved 1052 2017-03-28T01:47:30Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article<ref name="LoC">[Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.]</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this article show the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues,<ref name="LoC">[Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.]</ref>]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref name="LoC">[Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376.]</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182<br /> <br /> Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732536 Sandbox Reserved 1052 2017-03-28T01:45:47Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article<ref name="LoC">[Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.]</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this article show the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]<br /> <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref name="LoC">[Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376.]</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182<br /> <br /> Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376</div>

Mary Liggett https://proteopedia.org/index.php?title=File:Capture01.PNG&diff=2732535 File:Capture01.PNG 2017-03-28T01:40:55Z

<p>Mary Liggett: Val, Ala, and Gln Residues</p> <hr /> <div>Val, Ala, and Gln Residues</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732534 Sandbox Reserved 1052 2017-03-28T01:40:19Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article<ref name="LoC">[Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.]</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this article show the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change.<br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref name="LoC">[Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376.]</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182<br /> <br /> Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732533 Sandbox Reserved 1052 2017-03-28T01:23:03Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article<ref name="LoC">[Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.]</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this article show the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. <br /> The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref name="LoC">[Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376.]</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182<br /> <br /> Chakravorty D., Giedroc D., Merz K., Lee C., Wang B. (2012). Simulations of Allosteric Motions in the Zinc Sensor CzrA. JCAS 134 <br /> 3367-3376 <br /> <br /> Penella M., Shokes J., Cosper N., Scott R.,Giedroc D. (2002). Structural elements <br /> of metal selectivity in metal sensor proteins. PNAS 100:7 3713-3718.</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2732480 Sandbox Reserved 1052 2017-03-27T02:39:54Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article<ref name="LoC">[Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.]</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this articel show the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> for each mutation. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182<br /> <br /> Penella M., Shokes J., Cosper N., Scott R.,Giedroc D. (2002). Structural elements <br /> of metal selectivity in metal sensor proteins. PNAS 100:7 3713-3718.</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2728290 Sandbox Reserved 1052 2017-03-14T13:49:10Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; the Ka decresed by 11 and 160-fold respectively<ref name="LoC">[Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.]</ref> The previously mentioned Ser and His residues were found to bind the DNA with a similar affinity as the fully inhibited Zn<sup>2+</sup>. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> <br /> </StructureSection><br /> == References ==<br /> Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182<br /> <br /> Penella M., Shokes J., Cosper N., Scott R.,Giedroc D. (2002). Structural elements <br /> of metal selectivity in metal sensor proteins. PNAS 100:7 3713-3718.</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2728281 Sandbox Reserved 1052 2017-03-14T13:30:52Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> <br /> == Biological Function ==<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by replacing the Gln and Val with Ala residues and measuring the binding capacity; the Ka decresed by 11 and 160-fold respectively<ref name="LoC">[Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182.]</ref> The previously mentioned Ser and His residues were found to bind the DNA with a similar affinity as the fully inhibited Zn<sup>2+</sup>. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> == Other Ligands ==<br /> <br /> </StructureSection><br /> == References ==<br /> Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a <br /> paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 <br /> 18177-18182</div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2728258 Sandbox Reserved 1052 2017-03-14T13:03:46Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Biological Function ==<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the SER 54 and 57 along with HIS 58. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are VAL 42 and GLN 53. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> == Other Ligands ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2728255 Sandbox Reserved 1052 2017-03-14T13:00:34Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Biological Function ==<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/1'>main DNA binding residues</scene> have been found to be the SER 54 and 57 along with HIS 58. These residues are found in the N terminal of the R helix. The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are VAL 42 and GLN 53. <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> == Other Ligands ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=Sandbox_Reserved_1052&diff=2728246 Sandbox Reserved 1052 2017-03-14T12:47:16Z

<p>Mary Liggett: </p> <hr /> <div><Structure load='2KJB' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> ==CzrA==<br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Biological Function ==<br /> <br /> == Structural Overview ==<br /> <br /> == DNA Bound State ==<br /> The <scene name='69/694219/Serandhisresidues/1'>main DNA binding residues</scene> have been found to be the SER 54 and 57 along with HIS 58. These residues are found in the N terminal of the <br /> <br /> == Zinc Ligand(s) ==<br /> <br /> == Other Ligands ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=User:Mary_Liggett/Sandbox_1&diff=2716247 User:Mary Liggett/Sandbox 1 2017-02-28T14:43:13Z

<p>Mary Liggett: </p> <hr /> <div>=='''CzrA in Staphylococcus Aureus'''== <br /> ===''CzrA Mechanism''===<br /> ===''CzrA with Zn Bound''===<br /> [[Image:4GGG bio r 500.jpg|300px|right|thumb|Figure Legend]]<br /> <br /> <Structure load='4GGG' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> <br /> These are the <scene name='75/752355/Hydrophobic_residues/1'>Hydrophic Residues</scene> of CzrA. <br /> This is a default text for your page '''Mary Liggett/Sandbox 1'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/><br /> <br /> <scene name='75/752355/Hydrophobic_residues/1'>TextToBeDisplayed</scene></div>

Mary Liggett https://proteopedia.org/index.php?title=User:Mary_Liggett/Sandbox_1&diff=2716242 User:Mary Liggett/Sandbox 1 2017-02-28T14:39:33Z

<p>Mary Liggett: </p> <hr /> <div>=='''CzrA in Staphylococcus Aureus'''== <br /> ===''CzrA Mechanism''===<br /> ===''CzrA with Zn Bound''===<br /> [[Image:4GGG bio r 500.jpg|300px|right|thumb|Figure Legend]]<br /> <br /> <Structure load='4GGG' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> <StructureSection load='4GGG' size='340' side='right' caption='Hydrophic Residues of CzrA=''><br /> These are the <scene name='75/752355/Hydrophobic_residues/1'>Hydrophic Residues</scene> of CzrA. <br /> This is a default text for your page '''Mary Liggett/Sandbox 1'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/><br /> <br /> <scene name='75/752355/Hydrophobic_residues/1'>TextToBeDisplayed</scene></div>

Mary Liggett https://proteopedia.org/index.php?title=User:Mary_Liggett/Sandbox_1&diff=2716199 User:Mary Liggett/Sandbox 1 2017-02-28T14:09:35Z

<p>Mary Liggett: </p> <hr /> <div>=='''CzrA in Staphylococcus Aureus'''== <br /> ===''CzrA Mechanism''===<br /> ===''CzrA with Zn Bound''===<br /> [[Image:4GGG bio r 500.jpg|300px|right|thumb|Figure Legend]]<br /> <Structure load='4GGG' size='350' frame='true' align='left' caption='3D Representation of CzrA with Zn Bound' scene='Insert optional scene name here' /><br /> <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''><br /> This is a default text for your page '''Mary Liggett/Sandbox 1'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=User:Mary_Liggett/Sandbox_1&diff=2716191 User:Mary Liggett/Sandbox 1 2017-02-28T13:54:30Z

<p>Mary Liggett: </p> <hr /> <div>=='''CzrA in Staphylococcus Aureus'''== <br /> ===''CzrA Mechanism''===<br /> ===''CzrA with Zn Bound''===<br /> [[Image:4GGG bio r 500.jpg|300px|right|thumb|Figure Legend]]<br /> <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''><br /> This is a default text for your page '''Mary Liggett/Sandbox 1'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=File:4GGG_bio_r_500.jpg&diff=2716188 File:4GGG bio r 500.jpg 2017-02-28T13:48:57Z

<p>Mary Liggett: CzrA in the Zn bound state</p> <hr /> <div>CzrA in the Zn bound state</div>

Mary Liggett https://proteopedia.org/index.php?title=User:Mary_Liggett/Sandbox_1&diff=2716182 User:Mary Liggett/Sandbox 1 2017-02-28T13:39:05Z

<p>Mary Liggett: New page: =='''CzrA in Staphylococcus Aureus'''== ===''CzrA Mechanism''=== ===''CzrA with Zn Bound''=== <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' sc...</p> <hr /> <div>=='''CzrA in Staphylococcus Aureus'''== <br /> ===''CzrA Mechanism''===<br /> ===''CzrA with Zn Bound''===<br /> <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''><br /> This is a default text for your page '''Mary Liggett/Sandbox 1'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Mary Liggett https://proteopedia.org/index.php?title=User:Mary_Liggett&diff=2716162 User:Mary Liggett 2017-02-28T13:29:24Z

<p>Mary Liggett: </p> <hr /> <div>* Full Real Name: Mary Catherine Liggett<br /> <br /> * Position: Student<br /> <br /> * Institution (NO ABBREVIATIONS): Butler University <br /> <br /> * City, State/Province, Country: Indianapolis, IN, USA<br /> <br /> * Field of Expertise or Study: Biochemistry<br /> <br /> *[[User:Mary Liggett/Sandbox 1]]</div>

Mary Liggett https://proteopedia.org/index.php?title=User:Mary_Liggett&diff=2716159 User:Mary Liggett 2017-02-28T13:26:45Z

<p>Mary Liggett: </p> <hr /> <div>* Full Real Name: *[[User:Mary Catherine Liggett/ Sandbox 1]]<br /> <br /> * Position: Student<br /> <br /> * Institution (NO ABBREVIATIONS): Butler University <br /> <br /> * City, State/Province, Country: Indianapolis, IN, USA<br /> <br /> * Field of Expertise or Study: Biochemistry</div>

Mary Liggett https://proteopedia.org/index.php?title=User:Mary_Liggett&diff=2716158 User:Mary Liggett 2017-02-28T13:25:13Z

<p>Mary Liggett: </p> <hr /> <div>* Full Real Name: *[[User: Mary Catherine Liggett/ Sandbox 1]]<br /> <br /> * Position: Student<br /> <br /> * Institution (NO ABBREVIATIONS): Butler University <br /> <br /> * City, State/Province, Country: Indianapolis, IN, USA<br /> <br /> * Field of Expertise or Study: Biochemistry</div>

Mary Liggett