User:Tom Gluick/glutamine synthetase: Difference between revisions
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Glutamine synthetase appears to come in three major phylogenetically related classes of structures[http://pfam.sanger.ac.uk/family?acc=PF00120]. The GS-I class is found in many prokaryotes consists of twelve identical subunits arranged as two stacked and interlocking hexameric rings. The crystal structure of the Salmonella typhimurium enzyme has been solved and published in 1989, and its regulation is a subject of textbook discussion[http://www.ebi.ac.uk/pdbsum/2gls]. Briefly, the enzyme is regulated precisely via feedback inhibition by metabolic products such as glycine, alanine, carbamoyl phosphate, CTP and five others, and covalent modification via the adenylylation of tyrosyl residue. Crystal structures for Salmonella typhimurium glutamine synthetase illustrate adenylylated, unadenylylated, and ligand bound states (1lgr[http://www.proteopedia.org/wiki/index.php/1lgr], 2gls[http://www.proteopedia.org/wiki/index.php/2gls], 2lgo[http://www.proteopedia.org/wiki/index.php/2lgo], 1f1h[http://www.proteopedia.org/wiki/index.php/1f1h], 1fs2[http://www.proteopedia.org/wiki/index.php/1fs2] and 1fpy[http://www.proteopedia.org/wiki/index.php/1fpy]). Explaining how this enzyme works is the focus of your projects. <br/> <br/> | Glutamine synthetase appears to come in three major phylogenetically related classes of structures[http://pfam.sanger.ac.uk/family?acc=PF00120]. The GS-I class is found in many prokaryotes consists of twelve identical subunits arranged as two stacked and interlocking hexameric rings. The crystal structure of the Salmonella typhimurium enzyme has been solved and published in 1989, and its regulation is a subject of textbook discussion[http://www.ebi.ac.uk/pdbsum/2gls]. Briefly, the enzyme is regulated precisely via feedback inhibition by metabolic products such as glycine, alanine, carbamoyl phosphate, CTP and five others, and covalent modification via the adenylylation of tyrosyl residue. Crystal structures for Salmonella typhimurium glutamine synthetase illustrate adenylylated, unadenylylated, and ligand bound states (1lgr[http://www.proteopedia.org/wiki/index.php/1lgr], 2gls[http://www.proteopedia.org/wiki/index.php/2gls], 2lgo[http://www.proteopedia.org/wiki/index.php/2lgo], 1f1h[http://www.proteopedia.org/wiki/index.php/1f1h], 1fs2[http://www.proteopedia.org/wiki/index.php/1fs2] and 1fpy[http://www.proteopedia.org/wiki/index.php/1fpy]). Explaining how this enzyme works is the focus of your projects. <br/> <br/> | ||
The human enzyme is classed as GS-II. The quaternary structure consists of ten identical subunits arranged as two pentameric rings stacked atop each other [http://www.ebi.ac.uk/pdbsum/2d3b | The human enzyme is classed as GS-II. The quaternary structure consists of ten identical subunits arranged as two pentameric rings stacked atop each other [http://www.ebi.ac.uk/pdbsum/2d3b] as shown in the Java Applet on the right hand side of the page. It is activated by Mn and several other divalent metal cations and α-keotglutarate; the enzyme is inhibited by methionine sulfoxime, glycine and carbamoyl phosphate.<ref>Haussinger, D., & Schleiss, F., Glutamine metabolism and signaling in the liver, Forntiers in Bioscience 2007, 12, 371-391.</ref> <br/> <br/> | ||
Glutamine synthetase is a key component in controlling ammonia concentrations, maintaining nitrogen balance among organs, acid-base homeostasis, gene regulation and signaling.<ref> Curi, R Glutamine, gene expression, and cell function. Frontiers in Bioscience 2007 12: 344-357. <ref | Glutamine synthetase is a key component in controlling ammonia concentrations, maintaining nitrogen balance among organs, acid-base homeostasis, gene regulation and signaling.<ref> Curi, R., Glutamine, gene expression, and cell function. Frontiers in Bioscience 2007 12: 344-357. </ref> Glutamine homeostasis is maintained in part via regulating transcription and GS protein degradation. <ref> Labow, B. I., ''et.al.'', Mechanisms Governing the Expression of the Enzymes of Glutamine | ||
Metabolism—Glutaminase and Glutamine Synthetase. J. Nutr. 2001 131: 2467S–2474S.</ref>. | Metabolism—Glutaminase and Glutamine Synthetase. J. Nutr. 2001 131: 2467S–2474S.</ref>. | ||
In ureoteles, glutamine serves as a nontoxic shuttle of excess ammonia as its transported from tissues not capable of processing ammonia further through the blood to the liver where amide nitrogen eventually ends up in urea. For neurons in the brain to function normally, the excititory glutamate generated by the neuron is absorbed by the astrocyte and recycled to glutamine by GS.<ref> Albrecht, J. Glutamine in the central nervous system: function and dysfunction. Frontiers in Bioscience 2007 12:332-343. </ref> | In ureoteles, glutamine serves as a nontoxic shuttle of excess ammonia as its transported from tissues not capable of processing ammonia further through the blood to the liver where amide nitrogen eventually ends up in urea. For neurons in the brain to function normally, the excititory glutamate generated by the neuron is absorbed by the astrocyte and recycled to glutamine by GS.<ref> Albrecht, J., Glutamine in the central nervous system: function and dysfunction. Frontiers in Bioscience 2007 12:332-343. </ref> | ||
Disruptions in glutamine homeostasis brought about through cancer, trauma or HiV infection can lead to organ failure and death. Although extremely rare and only seen in the children of consnaguionoius couples, that defects in GLUL ( the gene encoding for gluatmine synthetase) causes a congenital disorder leading to brain malformation and death in neonates[http://ca.expasy.org/cgi-bin/niceprot.pl?P15104].<ref>Haberle, J. et.al., Congenital Glutamine Deficiency with Glutamine Synthetase Mutations. New Engl J Med 2003 353:1926-33.</ref> | Disruptions in glutamine homeostasis brought about through cancer, trauma or HiV infection can lead to organ failure and death. Although extremely rare and only seen in the children of consnaguionoius couples, that defects in GLUL ( the gene encoding for gluatmine synthetase) causes a congenital disorder leading to brain malformation and death in neonates[http://ca.expasy.org/cgi-bin/niceprot.pl?P15104].<ref>Haberle, J., ''et.al.'', Congenital Glutamine Deficiency with Glutamine Synthetase Mutations. New Engl J Med 2003 353:1926-33.</ref> | ||
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Before you start the project, I suggest you view the video tutorial in [http://www.proteopedia.org/wiki/index.php/Proteopedia:Video_Guide] to provide you with the tools to get started and give you a great overview of the power of proteopedia. I also suggest that you be aware of scene authoring tools with detailed explanations link given in[http://proteopedia.org/wiki/index.php/Scene_authoring_tools] and the editing page access in Help. | Before you start the project, I suggest you view the video tutorial in [http://www.proteopedia.org/wiki/index.php/Proteopedia:Video_Guide] to provide you with the tools to get started and give you a great overview of the power of proteopedia. I also suggest that you be aware of scene authoring tools with detailed explanations link given in[http://proteopedia.org/wiki/index.php/Scene_authoring_tools] and the editing page access in Help. | ||
==References== | |||
<references/> | |||
=Preface to the Project: First Things First= | =Preface to the Project: First Things First= | ||
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1. '''Open Scene authoring tools''' ( scroll down-this allows you to load an image from the PDB or a previously saved scene). <br/> | 1. '''Open Scene authoring tools''' ( scroll down-this allows you to load an image from the PDB or a previously saved scene). <br/> | ||
2. '''Click on the Load Molecule Tab''' (one of the options. This one allows you to load the PDB file).<br/> | 2. '''Click on the Load Molecule Tab''' (one of the options. This one allows you to load the PDB file).<br/> | ||
3. '''Type in 2qc8'''( | 3. '''Type in 2qc8'''(the combination of four letters or numbers signifies the file containing the coordinates to describe a crystal structure of human glutamine synthetase.).<br/> | ||
4. '''Click on Selections Tab''' (this tab will bring up a new window that will let you select all, none or parts of the structure).<br/> | 4. '''Click on Selections Tab''' (this tab will bring up a new window that will let you select all, none or parts of the structure).<br/> | ||
5. '''Click on Select None''' ( this sets it up so you want to add to a selection) Select all, you use this to remove stuff from selection.<br/> | 5. '''Click on Select None''' ( this sets it up so you want to add to a selection) Select all, you use this to remove stuff from selection.<br/> | ||
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1. Load 2qc8 | 1. '''Load''' 2qc8 | ||
2. Click select none under selection tabs. Limit the amino acid residues and ligands to chain A. Type in the amino acid residues that make hydrogen or electrostatic contacts with the ligands; do not include Mn at the moment. Obtain this information from the PSBsum webpage.<br/> | 2. '''Click''' select none under selection tabs. Limit the amino acid residues and ligands to chain A. Type in the amino acid residues that make hydrogen or electrostatic contacts with the ligands; do not include Mn at the moment. Obtain this information from the PSBsum webpage.<br/> | ||
3. Invert the selection, hide the inverted selection, and invert again.<br/> | 3. '''Invert''' the selection, hide the inverted selection, and invert again.<br/> | ||
4. | 4. ''' Center and zoom'''.<br/> | ||
5. Convert the selection structure from cartoon to wireframe.<br/> | 5. '''Convert''' the selection structure from cartoon to wireframe.<br/> | ||
6. Set the colors to CPK.<br/> | 6. '''Set''' the colors to CPK.<br/> | ||
7. Now select the three Mn in Chain A, use the halos to make sure you only select Mn. You might want to select none first. <br/> | 7. Now '''select''' the three Mn in Chain A, use the halos to make sure you only select Mn. You might want to select none first. <br/> | ||
8. Set the spacefill at 50% and set representation.<br/>[[Image:active_site_still.png]] | 8. '''Set''' the spacefill at 50% and set representation.<br/>[[Image:active_site_still.png]] | ||
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If you scroll up to the original JMOL applet, and click on ADP, you will notice that the image fades allowing the ligand to become prominent as it zooms in and out while the image spins. | If you scroll up to the original JMOL applet, and click on ADP, you will notice that the image fades allowing the ligand to become prominent as it zooms in and out while the image spins. To zoom you just need to hold down shift and then drag the molecule up and down to Zoom in the Scene Authoring Tools. <applet load='2qc8' size='300' frame='true' align='right' caption='JMOL Console Practice Applet' />This is fine, but to have precise control of the zoom use the JMOL commands or the Zoom command in the JMOL frank, the JMOL on the lower lefthand side of the applet. Unfortunately at this time we can only zoom in using JMOL commands in proteopedia. ( you can however, zoom in and zoom out using the commands in the console and see the results of your mastery of the command language in the applet, but when the scene is saved only the final state is saved, a bummer for now.) | ||
In the following paragraphs I will illustrate how to use several commands in JMOL which avoid some steps in the SAT. The commands i will illustrate are zoomto, select, restrict, color, translucent and... . | In the following paragraphs I will illustrate how to use several commands in JMOL which avoid some steps in the SAT. The commands i will illustrate are zoomto, select, restrict, color, translucent and... . | ||
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VII. Conclusion<br/> | VII. Conclusion<br/> | ||
=== | ===Assignments <ref>PDB codes for the assignment are listed in the introduction. For instance, PDB code for 1lgr may be useful in assignment 10.</ref>=== | ||
<span style="color:red">'''Assignment 1: IIA. Quaternary Structure:'''</span> Provide an overview of the quaternary structure of ''Salmonella typhimurium'' Glutamine Synthetase. Your explanation ought to include stoichiometry, how the monomers are joined to from the quaternary structure, a clever way to show the symmetry, an indication of the active site location.<ref name="review">Eisenberg, D., et.al., Structure-function relationships of glutamine synthetases, Biochim Biophys Acta 2000: 1477, 122-145.</ref><br/> | |||
<span style="color:red">'''Assignment 2: IIB. Tertiary Structure:''' </span> Map the two Pfam domains in the GS; Include in your discussion what Pfam database records<ref>Bateman, ''et.al.,''The Pfam Protein Families Database, Nucl Acids Res, 2000: 28, D263-D266;Bateman, ''et.al.,''The Pfam Protein Families Database, Nucl Acids Res, 2004: 32, D138-D141 </ref>; Define the role of each domain in the protiein. You can use the viewers provided in the Pfam site, but I had some difficulties with all three; JMOL did not load the molecule properly; Astex, never did load the molecule, and SPICE did, but the viewer is complicated to operate. Your experience may be different. Show the general design of the active site; reference 1 contains useful material<ref name="review"/>.<br/> | |||
<span style="color:red">'''Assignment 3: IIB. Tertiary Structure:'''</span> Map the two CATH domains in the GS; Include in your discussion what CATH database records<ref>Pearl, F., ''et.al.''The CATH Domain Structure Database and related resources Gene3D and DHS provide comprehensive domain family information for genome analysis, Nucl. Acids Res. 2005 22: D247-D252</ref>; explain the significance of each domain. In your discussion show the features of the CATH architecture and topology that provides them with the designation. As a hint you may want to use the Jena Library Jmol[http://www.imb-jena.de/IMAGE.html], which can be access through PDBsum[http://www.ebi.ac.uk/pdbsum/] to assist you in mapping the CATH domains. The RSCB site may not list the domains correctly.[http://www.rcsb.org/pdb/home/home.do] <ref name="review"/><br/> | |||
<span style="color: | <span style="color:red">'''Assignment 4: IIB: Tertiary Structure:''' </span> Map polar/nonpolar or charged and uncharged regions of GS monomers. Use space filling model. Map where polar and nonpolar residues are located in the protein. Comment on significance.<ref name="struct">Yamashita, M. M., ''et.al.,'' Refined Atolnic Model of Glutamine Synthetase at 3.5 A Resolution, J Biol Chem 1989 264: 17681-17690.</ref> | ||
<span style="color: | <span style="color:red">'''Assignment 5: IIC: Secondary Structure:''' </span> Dissect a monomer into secondary structure elements. You may want to include a wiring diagram or use the wiring diagram in PDBsum[http://www.ebi.ac.uk/pdbsum/] as an aid in presenting. Briefly comment on the significance of the elements when required.<ref name="struct"/> | ||
<span style="color: | <span style="color:red">'''Assignment 6: IID: Primary Structure:'''</span> Map the conserved residues on an individual subunit. Comment on the significance of the conservation using applets or images to enhance discussion of the topic. See discussion of Wiki Colors in Help: Editing to be used to enhanced your presentation. <ref name="review"/> <br/> | ||
<span style="color: | <span style="color:red">'''Assignment 7: IIIA: β-loop:'''</span> Map the interactions stabilizing quaternary structure. Explain how these interactions contribute to quaternary structure stability. <ref name="review"/><ref name="struct"/><br/> | ||
<span style="color: | <span style="color:red">'''Assignment 8: IIIB: Central-loop:''' </span> Map the interactions involving the central loop that stabilize quaternary structure. Explain how these interactions contribute to quaternary structure stability.<ref name="review"/><ref name="struct"/><br/> | ||
<span style="color: | <span style="color:red">'''Assignment 9: IIIC: Helical thong:''' </span> Map the interactions involving the helical thong that stabilize quaternary structure. Explain how these interactions contribute to quaternary structure stability.<ref name="review"/><ref name="struct"/><br/> | ||
<span style="color: | <span style="color:red">'''Assignment 10: IVA: ATP Binding site:''' </span> Map the ATP binding site; indicate which residues stabilize ATP binding; indicate which residues are important for activity and how they contribute to catalysis. <ref name="review"/><ref name="struct"/><ref name="atp">Liaw, S-H., Interactions of Nucleotides with Fully Unadenylylated Glutamine Synthetase from Salmonella typhimurium, Biochemistry 1994, 33: 11184-11188 </ref><br/> | ||
<span style="color: | <span style="color:red">'''Assignment 11: IVB: Glu binding site:''' </span> Map out the Glu binding site and explain how Glu binds, you will need to consider the inhibitor phosphothricin as a good analog of glutamate.<ref>Gill, H & Eisenberg, D., Biochemistry 2001 40: 1903-1912</ref> Indicate which amino acid residues are important in stabilizing Glu in the binding site. Indicate which residues are involved in chemistry.<ref name="review"/><ref name="myco">Krajewski, W. W., ''et.al.,''Structure of ''Mycobacterium tuberculosis'' glutamine synthetase in complex with a transition-state mimic provides functional insights, Proc Natl Acad Sci 2005, 102; 10499-10504.</ref><br/> | ||
<span style="color: | <span style="color:red">'''Assignment 12: IVC: Ammonium Binding Site:''' </span> Map the Ammonium binding site and explain how it contributes to cataysis.<ref name="NH4">Liaw, S-H, ''et.al.,''Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site Protein Sci. 1995 4: 2358-2365</ref><br/> | ||
<span style="color: | <span style="color:red">'''Assignment 13: IVD: Inhibitors:'''</span> Explain how a competitor inhibitor binds to the active site. <ref name="review"/><ref name="inhib">Liaw, S-H., ''et.al.,''Feedback inhibition of fully unadenylylated glutamine synthetase from ''Salmonella typhimurium'' by glycine, alanine, and serine, Proc Natl Acad Sci 1993 90: 4996-5000.</ref><br/> | ||
<span style="color:red">'''Assignment 14: VA:''' </span> Transition State Analogs: Map the transition state in ''Mycobacterium tuberculosis'' crystal structure. Show the important interactions involved in chemical steps directly.<ref name="myco"/> <br/> | |||
<span style="color:red">'''Assignment 15: VIA:''' </span> Map the Adenylylation site on the tertiary and quaternary structure of GS with an explanation of how it happens and how the covalent modification affects enzyme activity.<ref name="review"/> <br/> | |||
<span style="color:red">'''Assignment 15: VIB:''' </span> Map the ADP-ribosylation site on the tertiary and quaternary structure of GS with an explanation of how it happens and how the covalent modification affects enzyme activity.<ref name="review"/><br/> | |||
<span style="color:red">'''Instructor Project: I and II:'''</span> The introduction and the conclusion. | |||
== Examples of Student Projects == | |||
<br/> | |||
A few comments on the assignment are below each presentation.<br/> | |||
To see an example of <span style="color:red">'''Assignment 3: IIB. Tertiary Structure:'''</span>[[/Assignment 3]]<br/> | |||
To see an example of <span style="color:red">'''Assignment 8: IIIB. Central Loop:'''</span>[[/Assignment 8]]<br/> | |||
To see an example of <span style="color:red">'''Assignment 1: IIA. Quaternary Structure:'''</span>[[/Assignment 1]]<br/> | |||
== Overall Assessment and Advice to Others Wishing to Design Assignments in Proteopedia == | |||
I taught a one-semester biochemistry course, where I focused upon structures, enzymes, kinetics. regulation and pathways. Topics related to the central dogma were not discussed. <br/> | |||
Procedure: Students chose three topics from the list above and were assigned one of their choices for which they received one point. Students then registered at Proteopedia and completed the assignment teaching them to become comfortable with SAT. Completing the assignment earned the students 5 points. Students then submitted a rough draft. Students were evaluated on rough drafts and were required to meet with the instructor for mid course adjustments. The rough draft included all test and included place holders for WIKI scripts. Students could earn a maximum of nine points for the rough draft. Students submitted the final draft. If I felt more work was required, I gave the students more time to complete the task. Between the time of the rough and final draft students were allowed to ask to have their sites evaluated without being graded. Students could earn 10 points for the final draft. Total points: 25 points.<br/> | |||
Grading was lenient since students were beta-testers.<br/> | |||
What I learned: The SAT is simple enough for students to master, a testament to those who designed the tools. Most students were able to muster a reasonable project; at least one project was top notch; several were very good and one was a failure. Students can produce very elegant scripts, but most will not have the patience nor imagination to do so. | |||
=References= | |||
< | <references /> | ||