User:J. Shaun Lott/BIOSCI 203: Difference between revisions

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Q1: How many α-helices are there in this protein?

Q6: How many α-helices are there in this protein?

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Q2: How many β-strands make up the β-sheet?

Q7: How many β-strands make up the β-sheet?

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Q3: Is the topology of the β-sheet parallel, anti-parallel or mixed?

Q8: Is the topology of the β-sheet parallel, anti-parallel or mixed?

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hORF6 is an example of a 1-Cys Prx, and central to its enzymatic activity is its ability to maintain the active site cysteine residue in the charged (deprotonated) form of a thiolate ion (-S-) rather than the usual uncharged (protonated) sulfhydryl form (-SH).

Use the Henderson-Hasselbach equation to answer the questions below. The pKa of the –SH group of free cysteine is 8.5. The cytosol of human cells is normally at pH 7.3. The pKa of the active site cysteine in a 1-Cys Prx enzyme has been measured at 6.0.

Use the Henderson-Hasselbach equation to answer the questions below.

(The pKa of the –SH group of free cysteine is 8.5. The cytosol of human cells is normally at pH 7.3. The pKa of the active site cysteine in a 1-Cys Prx enzyme has been measured at 6.0.)

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Q4: What would be the % ionization of the –SH group of free cysteine in the cytosol?

Q9: What would be the % ionization of the –SH group of free cysteine in the cytosol?

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Q5: What would be the % ionization of the –SH group of the Prx active site cysteine in the cytosol?

Q10: What would be the % ionization of the –SH group of the Prx active site cysteine in the cytosol?

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Now let’s look at the structure to see if we can identify what features of the ~~hORF~~ structure may influence the ability of the active site cysteine to ionize.

Now let’s look more closely at the hORF structure to see if we can identify what local features of the protein structure may influence the ability of the active site cysteine to ionize.

<scene name='User:J._Shaun_Lott/BIOSCI_203/Active_site_cys/1'>Here</scene> is a close-up view of the active site cysteine residue (Cys47) shown in 'ball and stick' representation. We want to know what other amino acid sidechains are close enough to influence the ionization of the cysteine sulfhydyl (-SH) group. If we now <scene name='User:J._Shaun_Lott/BIOSCI_203/Active_site_cys_4a/3'>show</scene> other amino acid sidechains that have atoms within 4Å of the sulfur atom of Cys47, we can start to see what properties of these residues might be important in the ionization of Cys47.

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Q11: Which residues have side chains close to Cys47? (Hint: try mousing over each of the displayed sidechains to identify them.)

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Q12: What feature of the local environment around Cys74 might stabilize the existence of a thiolate ion?

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~~<applet load="HORF6.pdb" size="500" color="white" frame="true" align="left" caption="Active site" />~~

@@ Line 17: / Line 17: @@
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-Q1: How many α-helices are there in this protein?
+Q6: How many α-helices are there in this protein?
 ----
-Q2: How many β-strands make up the β-sheet?
+Q7: How many β-strands make up the β-sheet?
 ----
-Q3: Is the topology of the β-sheet parallel, anti-parallel or mixed?
+Q8: Is the topology of the β-sheet parallel, anti-parallel or mixed?
 ----
@@ Line 33: / Line 33: @@
 hORF6 is an example of a 1-Cys Prx, and central to its enzymatic activity is its ability to maintain the active site cysteine residue in the charged (deprotonated) form of a thiolate ion (-S<sup>-</sup>) rather than the usual uncharged (protonated) sulfhydryl form (-SH).
-Use the Henderson-Hasselbach equation to answer the questions below. The p<i>K</i><sub>a</sub> of the –SH group of free cysteine is 8.5. The cytosol of human cells is normally at pH 7.3. The p<i>K</i><sub>a</sub> of the active site cysteine in a 1-Cys Prx enzyme has been measured at 6.0.
+Use the Henderson-Hasselbach equation to answer the questions below.
+(The p<i>K</i><sub>a</sub> of the –SH group of free cysteine is 8.5. The cytosol of human cells is normally at pH 7.3. The p<i>K</i><sub>a</sub> of the active site cysteine in a 1-Cys Prx enzyme has been measured at 6.0.)
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-Q4: What would be the % ionization of the –SH group of free cysteine in the cytosol?
+Q9: What would be the % ionization of the –SH group of free cysteine in the cytosol?
 ----
-Q5: What would be the % ionization of the –SH group of the Prx active site cysteine in the cytosol?
+Q10: What would be the % ionization of the –SH group of the Prx active site cysteine in the cytosol?
 ----
-Now let’s look at the structure to see if we can identify what features of the hORF structure may influence the ability of the active site cysteine to ionize.
+Now let’s look more closely at the hORF structure to see if we can identify what local features of the protein structure may influence the ability of the active site cysteine to ionize.
+<applet load="HORF6.pdb" size="500" color="white" frame="true" align="left" caption="Active site" />
+<scene name='User:J._Shaun_Lott/BIOSCI_203/Active_site_cys/1'>Here</scene> is a close-up view of the active site cysteine residue (Cys47) shown in 'ball and stick' representation. We want to know what other amino acid sidechains are close enough to influence the ionization of the cysteine sulfhydyl (-SH) group. If we now <scene name='User:J._Shaun_Lott/BIOSCI_203/Active_site_cys_4a/3'>show</scene> other amino acid sidechains that have atoms within 4Å of the sulfur atom of Cys47, we can start to see what properties of these residues might be important in the ionization of Cys47.
+----
+Q11: Which residues have side chains close to Cys47? (Hint: try mousing over each of the displayed sidechains to identify them.)
+----
+Q12: What feature of the local environment around Cys74 might stabilize the existence of a thiolate ion?
+----
+----
-<applet load="HORF6.pdb" size="500" color="white" frame="true" align="left" caption="Active site" />
 {{Clear}}