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

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~~<applet load="HORF6.pdb" size="500" color="white" frame="true" align="right" caption="hORF6" />~~

~~== '''BIOSCI 203 Lab 2 - Protein structure''' ==~~

== '''BIOSCI 203 Lab 1 - Protein structure''' ==

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On the right-hand side of this page is a view of a protein structure shown using the Jmol viewer within [http://proteopedia.org/wiki/index.php/Main_Page Proteopedia]. [http://wiki.jmol.org:81/index.php/Main_Page Jmol] is an easy way to view protein structures. Use the left mouse button to rotate the protein model, the middle mouse button or scroll wheel to zoom in and out (option-click on a Mac), and the right mouse button for more options and information (control-click on a Mac). Try this out for yourself now ==>

The [http://www.rcsb.org/pdb/explore/explore.do?structureId=1PRX protein structure] we will be looking at in this part of the lab is the N-terminal domain of the human 1-Cys peroxidase enzyme [http://www.nature.com.ezproxy.auckland.ac.nz/nsmb/journal/v5/n5/abs/nsb0598-400.html hORF6]. Click

The [http://www.rcsb.org/pdb/explore/explore.do?structureId=1PRX protein][http://www.proteopedia.org/wiki/index.php/1prx structure] we will be looking at in this part of the lab is the N-terminal domain of the human 1-Cys peroxidase enzyme [http://www.nature.com.ezproxy.auckland.ac.nz/nsmb/journal/v5/n5/abs/nsb0598-400.html hORF6]. Click

<scene name='User:J._Shaun_Lott/BIOSCI_203/All_atoms_view/1'>here</scene> for an 'all atoms' view of the protein. This will show us all the atoms (except for the hydrogens) coloured by the 'CPK' colour scheme we talked about in lectures - blue for nitrogen atoms, red for oxygen atoms, grey for carbon atoms and yellow for sulfur atoms. Pretty hard to see what's going on, isn't it?

We can simplify things by just showing a cartoon that traces the path of the amino acid backbone; α-helices are shown as coils, and β-strands as arrows pointing in the direction of the C-terminus of the protein. <scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_rainbow/2'>Here</scene> is a version with the cartoon coloured from blue at the N-terminus to red at the C-terminus. <scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_elements/1'>Here </scene>is a version with the β-strands coloured yellow and the α-helices coloured pink.

We can simplify things by just showing a cartoon that traces the path of the amino acid backbone; α-helices are shown as coils, and β-strands as arrows pointing in the direction of the C-terminus of the protein. <scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_rainbow/3'>Here</scene> is a version with the cartoon coloured from blue at the N-terminus to red at the C-terminus. <scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_elements/2'>Here </scene>is a version with the β-strands coloured yellow and the α-helices coloured pink.

It can help to see the

<scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_hbonds/1'>backbone hydrogen bonds</scene> that define the secondary structure elements, and sometimes this is clearer shown as a <scene name='User:J._Shaun_Lott/BIOSCI_203/Backbone_hbonds/1'>backbone trace</scene> which just shows links between the Cα atoms, rather than a ribbon diagram.

<scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_hbonds/3'>backbone hydrogen bonds</scene> that define the secondary structure elements, and sometimes this is clearer shown as a <scene name='User:J._Shaun_Lott/BIOSCI_203/Backbone_hbonds/2'>backbone trace</scene> which just shows links between the Cα atoms, rather than a ribbon diagram.

----

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

'''Q7: How many α-helices are there in this protein?'''

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

'''Q8: 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?

'''Q9: 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.

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

(The pKa of the –SH group of free cysteine is 8.35. The cytosol of human cells is normally at pH 7.35 The pKa of the active site cysteine in a 1-Cys Prx enzyme has been measured at 6.2.)

----

'''Q10: What would be the % ionization of the –SH group of free cysteine in the cytosol?'''

----

'''Q11: What would be the % ionization of the –SH group of the Prx active site cysteine in the cytosol?'''

----

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/2'>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/5'>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.

----

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

'''Q12: Give the name and residue number for each of the amino acid residues that have side chains within 4Å of the sulfur atom in Cys47. (Hint: try mousing over each of the displayed sidechains to identify them.)'''

----

~~The pKa~~ of the ~~active site cysteine in~~ a ~~1-Cys Prx enzyme has been measured at 6.0.~~

'''Q13: What feature of the local environment around Cys47 might stabilize the existence of a thiolate ion?'''

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~~Q5: 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.~~

@@ Line 1: / Line 1: @@
-<applet load="HORF6.pdb" size="500" color="white" frame="true" align="right" caption="hORF6" />
+{{Template:Protected page banner}}
-== '''BIOSCI 203 Lab 2 - Protein structure''' ==
+== '''BIOSCI 203 Lab 1 - Protein structure''' ==
+<StructureSection load="HORF6.pdb" size="500" color="white" frame="true" align="right" caption="hORF6 (PDB ID=1PRX)" />
 ----
@@ Line 8: / Line 9: @@
 On the right-hand side of this page is a view of a protein structure shown using the Jmol viewer within [http://proteopedia.org/wiki/index.php/Main_Page Proteopedia]. [http://wiki.jmol.org:81/index.php/Main_Page Jmol] is an easy way to view protein structures. Use the left mouse button to rotate the protein model, the middle mouse button or scroll wheel to zoom in and out (option-click on a Mac), and the right mouse button for more options and information (control-click on a Mac). Try this out for yourself now ==>
-The [http://www.rcsb.org/pdb/explore/explore.do?structureId=1PRX protein structure] we will be looking at in this part of the lab is the N-terminal domain of the human 1-Cys peroxidase enzyme [http://www.nature.com.ezproxy.auckland.ac.nz/nsmb/journal/v5/n5/abs/nsb0598-400.html hORF6]. Click
+The [http://www.rcsb.org/pdb/explore/explore.do?structureId=1PRX protein][http://www.proteopedia.org/wiki/index.php/1prx structure] we will be looking at in this part of the lab is the N-terminal domain of the human 1-Cys peroxidase enzyme [http://www.nature.com.ezproxy.auckland.ac.nz/nsmb/journal/v5/n5/abs/nsb0598-400.html hORF6]. Click
 <scene name='User:J._Shaun_Lott/BIOSCI_203/All_atoms_view/1'>here</scene> for an 'all atoms' view of the protein. This will show us all the atoms (except for the hydrogens) coloured by the 'CPK' colour scheme we talked about in lectures - blue for nitrogen atoms, red for oxygen atoms, grey for carbon atoms and yellow for sulfur atoms. Pretty hard to see what's going on, isn't it?
-We can simplify things by just showing a cartoon that traces the path of the amino acid backbone; α-helices are shown as coils, and β-strands as arrows pointing in the direction of the C-terminus of the protein. <scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_rainbow/2'>Here</scene> is a version with the cartoon coloured from blue at the N-terminus to red at the C-terminus. <scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_elements/1'>Here </scene>is a version with the β-strands coloured yellow and the α-helices coloured pink.
+We can simplify things by just showing a cartoon that traces the path of the amino acid backbone; α-helices are shown as coils, and β-strands as arrows pointing in the direction of the C-terminus of the protein. <scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_rainbow/3'>Here</scene> is a version with the cartoon coloured from blue at the N-terminus to red at the C-terminus. <scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_elements/2'>Here </scene>is a version with the β-strands coloured yellow and the α-helices coloured pink.
 It can help to see the
-<scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_hbonds/1'>backbone hydrogen bonds</scene> that define the secondary structure elements, and sometimes this is clearer shown as a  <scene name='User:J._Shaun_Lott/BIOSCI_203/Backbone_hbonds/1'>backbone trace</scene> which just shows links between the Cα atoms, rather than a ribbon diagram.
+<scene name='User:J._Shaun_Lott/BIOSCI_203/Ss_hbonds/3'>backbone hydrogen bonds</scene> that define the secondary structure elements, and sometimes this is clearer shown as a  <scene name='User:J._Shaun_Lott/BIOSCI_203/Backbone_hbonds/2'>backbone trace</scene> which just shows links between the Cα atoms, rather than a ribbon diagram.
 ----
-Q1: How many α-helices are there in this protein?
+'''Q7: How many α-helices are there in this protein?'''
 ----
-Q2: How many β-strands make up the β-sheet?
+'''Q8: How many β-strands make up the β-sheet?'''
 ----
-Q3: Is the topology of the β-sheet parallel, anti-parallel or mixed?
+'''Q9: Is the topology of the β-sheet parallel, anti-parallel or mixed?'''
 ----
@@ Line 33: / Line 35: @@
 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.
+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 8.35. The cytosol of human cells is normally at pH 7.35 The p<i>K</i><sub>a</sub> of the active site cysteine in a 1-Cys Prx enzyme has been measured at 6.2.)
+----
+'''Q10: What would be the % ionization of the –SH group of free cysteine in the cytosol?'''
+----
+'''Q11: What would be the % ionization of the –SH group of the Prx active site cysteine in the cytosol?'''
+----
+----
+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/2'>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/5'>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.
 ----
-Q4: What would be the % ionization of the –SH group of free cysteine in the cytosol?
+'''Q12: Give the name and residue number for each of the amino acid residues that have side chains within 4Å of the sulfur atom in Cys47. (Hint: try mousing over each of the displayed sidechains to identify them.)'''
 ----
-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.
+'''Q13: What feature of the local environment around Cys47 might stabilize the existence of a thiolate ion?'''
 ----
-Q5: 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.
+{{Clear}}