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| [[Image:1qud.jpg|left|200px]]<br /><applet load="1qud" size="350" color="white" frame="true" align="right" spinBox="true"
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| caption="1qud, resolution 1.75Å" />
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| '''L99G MUTANT OF T4 LYSOZYME'''<br />
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| ==Overview== | | ==L99G MUTANT OF T4 LYSOZYME== |
| The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.
| | <StructureSection load='1qud' size='340' side='right'caption='[[1qud]], [[Resolution|resolution]] 1.75Å' scene=''> |
| | == Structural highlights == |
| | <table><tr><td colspan='2'>[[1qud]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_virus_T4 Escherichia virus T4]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1QUD OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1QUD FirstGlance]. <br> |
| | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.75Å</td></tr> |
| | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=HEZ:HEXANE-1,6-DIOL'>HEZ</scene></td></tr> |
| | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1qud FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1qud OCA], [https://pdbe.org/1qud PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1qud RCSB], [https://www.ebi.ac.uk/pdbsum/1qud PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1qud ProSAT]</span></td></tr> |
| | </table> |
| | == Function == |
| | [https://www.uniprot.org/uniprot/ENLYS_BPT4 ENLYS_BPT4] Endolysin with lysozyme activity that degrades host peptidoglycans and participates with the holin and spanin proteins in the sequential events which lead to the programmed host cell lysis releasing the mature viral particles. Once the holin has permeabilized the host cell membrane, the endolysin can reach the periplasm and break down the peptidoglycan layer.<ref>PMID:22389108</ref> |
| | == Evolutionary Conservation == |
| | [[Image:Consurf_key_small.gif|200px|right]] |
| | Check<jmol> |
| | <jmolCheckbox> |
| | <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/qu/1qud_consurf.spt"</scriptWhenChecked> |
| | <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> |
| | <text>to colour the structure by Evolutionary Conservation</text> |
| | </jmolCheckbox> |
| | </jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1qud ConSurf]. |
| | <div style="clear:both"></div> |
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| ==About this Structure== | | ==See Also== |
| 1QUD is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacteriophage_t4 Bacteriophage t4] with <scene name='pdbligand=CL:'>CL</scene> and <scene name='pdbligand=HEZ:'>HEZ</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Lysozyme Lysozyme], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.17 3.2.1.17] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1QUD OCA].
| | *[[Lysozyme 3D structures|Lysozyme 3D structures]] |
| | | == References == |
| ==Reference== | | <references/> |
| Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability., Wray JW, Baase WA, Lindstrom JD, Weaver LH, Poteete AR, Matthews BW, J Mol Biol. 1999 Oct 8;292(5):1111-20. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=10512706 10512706]
| | __TOC__ |
| [[Category: Bacteriophage t4]] | | </StructureSection> |
| [[Category: Lysozyme]]
| | [[Category: Escherichia virus T4]] |
| [[Category: Single protein]] | | [[Category: Large Structures]] |
| [[Category: Baase, W A.]] | | [[Category: Baase WA]] |
| [[Category: Lindstrom, J D.]] | | [[Category: Lindstrom JD]] |
| [[Category: Matthews, B W.]] | | [[Category: Matthews BW]] |
| [[Category: Poteete, A R.]] | | [[Category: Poteete AR]] |
| [[Category: Wray, J.]] | | [[Category: Wray J]] |
| [[Category: CL]]
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| [[Category: HEZ]]
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| [[Category: hydrolase]]
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| ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:43:50 2008''
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