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| | {{STRUCTURE_2p0x| PDB=2p0x | SCENE= }} | | {{STRUCTURE_2p0x| PDB=2p0x | SCENE= }} |
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| - | '''solution structure of a non-biological ATP-binding protein'''
| + | ===solution structure of a non-biological ATP-binding protein=== |
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| - | ==Overview==
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| - | 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.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_17583732}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 17583732 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_17583732}} |
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| | ==About this Structure== | | ==About this Structure== |
| - | Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2P0X OCA]. | + | Full experimental information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2P0X OCA]. |
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| | ==Reference== | | ==Reference== |
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| | [[Category: De novo protein]] | | [[Category: De novo protein]] |
| | [[Category: Treble clef zinc binding motif]] | | [[Category: Treble clef zinc binding motif]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 12:06:11 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 09:34:12 2008'' |
Revision as of 06:34, 28 July 2008
Template:STRUCTURE 2p0x
solution structure of a non-biological ATP-binding protein
Template:ABSTRACT PUBMED 17583732
About this Structure
Full experimental 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
Page seeded by OCA on Mon Jul 28 09:34:12 2008
