2p0x
From Proteopedia
(New page: 200px<br /><applet load="2p0x" size="350" color="white" frame="true" align="right" spinBox="true" caption="2p0x" /> '''solution structure of a non-biological ATP-b...) |
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==Overview== | ==Overview== | ||
| - | We present a structural and functional analysis of the evolutionary | + | We present a structural and functional analysis of the evolutionary optimization of a non-biological protein derived from a library of random amino acid sequences. A series of previously described in vitro selection experiments transformed a low-affinity ancestral sequence into a stably folded, high affinity ATP binding protein structure. While the evolutionarily optimized protein differs from its ancestral sequence through the accumulation of 12 amino acid mutations, the means by which those mutations enhance the stability and functionality of the protein were not well understood. We used a combination of mutagenesis, biochemistry, and NMR spectroscopy to investigate the structural and functional significance of each mutation. We solved the three-dimensional structure of the folding optimized protein by solution NMR, which revealed a fourth strand of the beta-sheet of the alpha/beta-fold that was not observed in an earlier crystallographic analysis of a less stable version of the protein. The structural rigidity of the newly identified beta-strand was confirmed by T1, T2, and heteronuclear nuclear Overhauser enhancement (NOE) measurements. Biochemical experiments were used to examine point mutations that revert the optimized protein back to the ancestral residue at each of the 12 sites. A combination of structural and functional data was then used to interpret the significance of each amino acid mutation. The enhanced ATP affinity was largely due to the emergence of a patch of positive charge density on the protein surface, while the increased solubility resulted from several mutations that increased the hydrophilicity of the protein surface, thereby decreasing protein aggregation. One mutation may stabilize the hydrophobic face of the beta-sheet. |
==About this Structure== | ==About this Structure== | ||
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==Reference== | ==Reference== | ||
| - | Structure and | + | Structure and evolutionary analysis of a non-biological ATP-binding protein., Mansy SS, Zhang J, Kummerle R, Nilsson M, Chou JJ, Szostak JW, Chaput JC, J Mol Biol. 2007 Aug 10;371(2):501-13. Epub 2007 May 26. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=17583732 17583732] |
[[Category: Protein complex]] | [[Category: Protein complex]] | ||
[[Category: Synthetic gene]] | [[Category: Synthetic gene]] | ||
| - | [[Category: Chaput, J | + | [[Category: Chaput, J C.]] |
| - | [[Category: Mansy, S | + | [[Category: Mansy, S S.]] |
| - | [[Category: Szostak, J | + | [[Category: Szostak, J W.]] |
[[Category: ATP]] | [[Category: ATP]] | ||
[[Category: ZN]] | [[Category: ZN]] | ||
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[[Category: de novo protein]] | [[Category: de novo protein]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 18:24:39 2008'' |
Revision as of 16:24, 21 February 2008
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solution structure of a non-biological ATP-binding protein
Overview
We present a structural and functional analysis of the evolutionary optimization of a non-biological protein derived from a library of random amino acid sequences. A series of previously described in vitro selection experiments transformed a low-affinity ancestral sequence into a stably folded, high affinity ATP binding protein structure. While the evolutionarily optimized protein differs from its ancestral sequence through the accumulation of 12 amino acid mutations, the means by which those mutations enhance the stability and functionality of the protein were not well understood. We used a combination of mutagenesis, biochemistry, and NMR spectroscopy to investigate the structural and functional significance of each mutation. We solved the three-dimensional structure of the folding optimized protein by solution NMR, which revealed a fourth strand of the beta-sheet of the alpha/beta-fold that was not observed in an earlier crystallographic analysis of a less stable version of the protein. The structural rigidity of the newly identified beta-strand was confirmed by T1, T2, and heteronuclear nuclear Overhauser enhancement (NOE) measurements. Biochemical experiments were used to examine point mutations that revert the optimized protein back to the ancestral residue at each of the 12 sites. A combination of structural and functional data was then used to interpret the significance of each amino acid mutation. The enhanced ATP affinity was largely due to the emergence of a patch of positive charge density on the protein surface, while the increased solubility resulted from several mutations that increased the hydrophilicity of the protein surface, thereby decreasing protein aggregation. One mutation may stabilize the hydrophobic face of the beta-sheet.
About this Structure
2P0X is a Protein complex structure of sequences from Synthetic gene with and as ligands. Full crystallographic information is available from OCA.
Reference
Structure and evolutionary analysis of a non-biological ATP-binding protein., Mansy SS, Zhang J, Kummerle R, Nilsson M, Chou JJ, Szostak JW, Chaput JC, J Mol Biol. 2007 Aug 10;371(2):501-13. Epub 2007 May 26. PMID:17583732
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