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[[ | ==Changing the determinants of protein stability from covalent to non-covalent interactions by in-vitro evolution: a structural and energetic analysis== | ||
<StructureSection load='3dgs' size='340' side='right' caption='[[3dgs]], [[Resolution|resolution]] 1.90Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[3dgs]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Enterobacteria_phage_fd Enterobacteria phage fd]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3DGS OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3DGS FirstGlance]. <br> | |||
</td></tr><tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">III ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=10864 Enterobacteria phage fd])</td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3dgs FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3dgs OCA], [http://www.rcsb.org/pdb/explore.do?structureId=3dgs RCSB], [http://www.ebi.ac.uk/pdbsum/3dgs PDBsum]</span></td></tr> | |||
</table> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
The three disulfide bonds of the gene-3-protein of the phage fd are essential for the conformational stability of this protein, and it unfolds when they are removed by reduction or mutation. Previously, we used an iterative in vitro selection strategy to generate a stable and functional form of the gene-3-protein without these disulfides. It yielded optimal replacements for the disulfide bonds as well as several stabilizing second-site mutations. The best selected variant showed a higher thermal stability compared with the disulfide-bonded wild-type protein. Here, we investigated the molecular basis of this strong stabilization by solving the crystal structure of this variant and by analyzing the contributions to the conformational stability of the selected mutations individually. They could mostly be explained by improved side-chain packing. The R29W substitution alone increased the midpoint of the thermal unfolding transition by 14 deg and the conformational stability by about 25 kJ mol(-1). This key mutation (i) removed a charged side chain that forms a buried salt bridge in the disulfide-containing wild-type protein, (ii) optimized the local packing with the residues that replace the C46-C53 disulfide and (iii) improved the domain interactions. Apparently, certain residues in proteins indeed play key roles for stability. | |||
Changing the determinants of protein stability from covalent to non-covalent interactions by in vitro evolution: a structural and energetic analysis.,Kather I, Jakob R, Dobbek H, Schmid FX J Mol Biol. 2008 Sep 12;381(4):1040-54. Epub 2008 Jul 2. PMID:18621056<ref>PMID:18621056</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
==See Also== | ==See Also== | ||
*[[G3p|G3p]] | *[[G3p|G3p]] | ||
== References == | |||
== | <references/> | ||
< | __TOC__ | ||
</StructureSection> | |||
[[Category: Enterobacteria phage fd]] | [[Category: Enterobacteria phage fd]] | ||
[[Category: Dobbek, H | [[Category: Dobbek, H]] | ||
[[Category: Jakob, R P | [[Category: Jakob, R P]] | ||
[[Category: Kather, I | [[Category: Kather, I]] | ||
[[Category: Schmid, F X | [[Category: Schmid, F X]] | ||
[[Category: Capsid protein]] | [[Category: Capsid protein]] | ||
[[Category: Dissulfide bond]] | [[Category: Dissulfide bond]] | ||
Revision as of 10:53, 12 November 2014
Changing the determinants of protein stability from covalent to non-covalent interactions by in-vitro evolution: a structural and energetic analysisChanging the determinants of protein stability from covalent to non-covalent interactions by in-vitro evolution: a structural and energetic analysis
Structural highlights
Publication Abstract from PubMedThe three disulfide bonds of the gene-3-protein of the phage fd are essential for the conformational stability of this protein, and it unfolds when they are removed by reduction or mutation. Previously, we used an iterative in vitro selection strategy to generate a stable and functional form of the gene-3-protein without these disulfides. It yielded optimal replacements for the disulfide bonds as well as several stabilizing second-site mutations. The best selected variant showed a higher thermal stability compared with the disulfide-bonded wild-type protein. Here, we investigated the molecular basis of this strong stabilization by solving the crystal structure of this variant and by analyzing the contributions to the conformational stability of the selected mutations individually. They could mostly be explained by improved side-chain packing. The R29W substitution alone increased the midpoint of the thermal unfolding transition by 14 deg and the conformational stability by about 25 kJ mol(-1). This key mutation (i) removed a charged side chain that forms a buried salt bridge in the disulfide-containing wild-type protein, (ii) optimized the local packing with the residues that replace the C46-C53 disulfide and (iii) improved the domain interactions. Apparently, certain residues in proteins indeed play key roles for stability. Changing the determinants of protein stability from covalent to non-covalent interactions by in vitro evolution: a structural and energetic analysis.,Kather I, Jakob R, Dobbek H, Schmid FX J Mol Biol. 2008 Sep 12;381(4):1040-54. Epub 2008 Jul 2. PMID:18621056[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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