3d2b: Difference between revisions
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< | ==Structure of 2D9, a thermostable mutant of Bacillus subtilis lipase obtained through directed evolution== | ||
<StructureSection load='3d2b' size='340' side='right'caption='[[3d2b]], [[Resolution|resolution]] 1.95Å' scene=''> | |||
You may | == Structural highlights == | ||
<table><tr><td colspan='2'>[[3d2b]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Bacillus_subtilis Bacillus subtilis]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3D2B OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3D2B 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]] 1.95Å</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=3d2b FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3d2b OCA], [https://pdbe.org/3d2b PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3d2b RCSB], [https://www.ebi.ac.uk/pdbsum/3d2b PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3d2b ProSAT]</span></td></tr> | ||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/ESTA_BACSU ESTA_BACSU] Active toward p-nitrophenyl esters and triacylglycerides with a marked preference for esters with C8 acyl groups.<ref>PMID:8396026</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/d2/3d2b_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=3d2b ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
In vitro evolution methods are now being routinely used to identify protein variants with novel and enhanced properties that are difficult to achieve using rational design. However, one of the limitations is in screening for beneficial mutants through several generations due to the occurrence of neutral/negative mutations occurring in the background of positive ones. While evolving a lipase in vitro from mesophilic Bacillus subtilis to generate thermostable variants, we have designed protocols that combine stringent three-tier testing, sequencing and stability assessments on the protein at the end of each generation. This strategy resulted in a total of six stabilizing mutations in just two generations with three mutations per generation. Each of the six mutants when evaluated individually contributed additively to thermostability. A combination of all of them resulted in the best variant that shows a remarkable 15 degrees C shift in melting temperature and a millionfold decrease in the thermal inactivation rate with only a marginal increase of 3 kcal mol(-1) in free energy of stabilization. Notably, in addition to the dramatic shift in optimum temperature by 20 degrees C, the activity has increased two- to fivefold in the temperature range 25-65 degrees C. High-resolution crystal structures of three of the mutants, each with 5 degrees increments in melting temperature, reveal the structural basis of these mutations in attaining higher thermostability. The structures highlight the importance of water-mediated ionic networks on the protein surface in imparting thermostability. Saturation mutagenesis at each of the six positions did not result in enhanced thermostability in almost all the cases, confirming the crucial role played by each mutation as revealed through the structural study. Overall, our study presents an efficient strategy that can be employed in directed evolution approaches employed for obtaining improved properties of proteins. | |||
Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight.,Ahmad S, Kamal MZ, Sankaranarayanan R, Rao NM J Mol Biol. 2008 Aug 29;381(2):324-40. Epub 2008 Jul 2. PMID:18599073<ref>PMID:18599073</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 3d2b" style="background-color:#fffaf0;"></div> | |||
== | |||
==See Also== | ==See Also== | ||
*[[Lipase]] | *[[Lipase 3D Structures|Lipase 3D Structures]] | ||
== References == | |||
== | <references/> | ||
< | __TOC__ | ||
</StructureSection> | |||
[[Category: Bacillus subtilis]] | [[Category: Bacillus subtilis]] | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: Kamal | [[Category: Kamal MZ]] | ||
[[Category: Sankaranarayanan | [[Category: Sankaranarayanan R]] | ||
Latest revision as of 22:20, 29 May 2024
Structure of 2D9, a thermostable mutant of Bacillus subtilis lipase obtained through directed evolutionStructure of 2D9, a thermostable mutant of Bacillus subtilis lipase obtained through directed evolution
Structural highlights
FunctionESTA_BACSU Active toward p-nitrophenyl esters and triacylglycerides with a marked preference for esters with C8 acyl groups.[1] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedIn vitro evolution methods are now being routinely used to identify protein variants with novel and enhanced properties that are difficult to achieve using rational design. However, one of the limitations is in screening for beneficial mutants through several generations due to the occurrence of neutral/negative mutations occurring in the background of positive ones. While evolving a lipase in vitro from mesophilic Bacillus subtilis to generate thermostable variants, we have designed protocols that combine stringent three-tier testing, sequencing and stability assessments on the protein at the end of each generation. This strategy resulted in a total of six stabilizing mutations in just two generations with three mutations per generation. Each of the six mutants when evaluated individually contributed additively to thermostability. A combination of all of them resulted in the best variant that shows a remarkable 15 degrees C shift in melting temperature and a millionfold decrease in the thermal inactivation rate with only a marginal increase of 3 kcal mol(-1) in free energy of stabilization. Notably, in addition to the dramatic shift in optimum temperature by 20 degrees C, the activity has increased two- to fivefold in the temperature range 25-65 degrees C. High-resolution crystal structures of three of the mutants, each with 5 degrees increments in melting temperature, reveal the structural basis of these mutations in attaining higher thermostability. The structures highlight the importance of water-mediated ionic networks on the protein surface in imparting thermostability. Saturation mutagenesis at each of the six positions did not result in enhanced thermostability in almost all the cases, confirming the crucial role played by each mutation as revealed through the structural study. Overall, our study presents an efficient strategy that can be employed in directed evolution approaches employed for obtaining improved properties of proteins. Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight.,Ahmad S, Kamal MZ, Sankaranarayanan R, Rao NM J Mol Biol. 2008 Aug 29;381(2):324-40. Epub 2008 Jul 2. PMID:18599073[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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