2fwq
From Proteopedia
(New page: 200px<br /><applet load="2fwq" size="450" color="white" frame="true" align="right" spinBox="true" caption="2fwq, resolution 1.40Å" /> '''Reduced enolate chro...) |
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| - | [[Image:2fwq.gif|left|200px]]<br /><applet load="2fwq" size=" | + | [[Image:2fwq.gif|left|200px]]<br /><applet load="2fwq" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="2fwq, resolution 1.40Å" /> | caption="2fwq, resolution 1.40Å" /> | ||
'''Reduced enolate chromophore intermediate for Y66H GFP variant'''<br /> | '''Reduced enolate chromophore intermediate for Y66H GFP variant'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The Aequorea victoria green fluorescent protein (GFP) creates a fluorophore from its component amino acids Ser65, Tyr66, and Gly67 through a remarkable post-translational modification, involving spontaneous peptide backbone cyclization, dehydration, and oxidation reactions. Here we test and extend the understanding of fluorophore biosynthesis by coupling chemical reduction and anaerobic methodologies with kinetic analyses and protein structure determination. Two high-resolution structures of dithionite-treated GFP variants reveal a previously uncharacterized enolate intermediate form of the chromophore that is viable in generating a fluorophore (t1 | + | The Aequorea victoria green fluorescent protein (GFP) creates a fluorophore from its component amino acids Ser65, Tyr66, and Gly67 through a remarkable post-translational modification, involving spontaneous peptide backbone cyclization, dehydration, and oxidation reactions. Here we test and extend the understanding of fluorophore biosynthesis by coupling chemical reduction and anaerobic methodologies with kinetic analyses and protein structure determination. Two high-resolution structures of dithionite-treated GFP variants reveal a previously uncharacterized enolate intermediate form of the chromophore that is viable in generating a fluorophore (t1/2 = 39 min-1) upon exposure to air. Isolation of this enolate intermediate will now allow specific probing of the rate-limiting oxidation step for fluorophore biosynthesis in GFP and its red fluorescent protein homologues. Such targeted characterizations may lead to the design of faster maturing proteins with enhanced applications in biotechnology and cell biology. Moreover, our results reveal how the GFP protein environment mimics enzyme systems, by stabilizing an otherwise high energy enolate intermediate to achieve its post-translational modification. |
==About this Structure== | ==About this Structure== | ||
| - | 2FWQ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Aequorea_victoria Aequorea victoria] with MG as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http:// | + | 2FWQ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Aequorea_victoria Aequorea victoria] with <scene name='pdbligand=MG:'>MG</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2FWQ OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Aequorea victoria]] | [[Category: Aequorea victoria]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Barondeau, D | + | [[Category: Barondeau, D P.]] |
| - | [[Category: Getzoff, E | + | [[Category: Getzoff, E D.]] |
| - | [[Category: Tainer, J | + | [[Category: Tainer, J A.]] |
[[Category: MG]] | [[Category: MG]] | ||
[[Category: beta barrel]] | [[Category: beta barrel]] | ||
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[[Category: intermediate]] | [[Category: intermediate]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:25:50 2008'' |
Revision as of 15:25, 21 February 2008
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Reduced enolate chromophore intermediate for Y66H GFP variant
Overview
The Aequorea victoria green fluorescent protein (GFP) creates a fluorophore from its component amino acids Ser65, Tyr66, and Gly67 through a remarkable post-translational modification, involving spontaneous peptide backbone cyclization, dehydration, and oxidation reactions. Here we test and extend the understanding of fluorophore biosynthesis by coupling chemical reduction and anaerobic methodologies with kinetic analyses and protein structure determination. Two high-resolution structures of dithionite-treated GFP variants reveal a previously uncharacterized enolate intermediate form of the chromophore that is viable in generating a fluorophore (t1/2 = 39 min-1) upon exposure to air. Isolation of this enolate intermediate will now allow specific probing of the rate-limiting oxidation step for fluorophore biosynthesis in GFP and its red fluorescent protein homologues. Such targeted characterizations may lead to the design of faster maturing proteins with enhanced applications in biotechnology and cell biology. Moreover, our results reveal how the GFP protein environment mimics enzyme systems, by stabilizing an otherwise high energy enolate intermediate to achieve its post-translational modification.
About this Structure
2FWQ is a Single protein structure of sequence from Aequorea victoria with as ligand. Full crystallographic information is available from OCA.
Reference
Structural evidence for an enolate intermediate in GFP fluorophore biosynthesis., Barondeau DP, Tainer JA, Getzoff ED, J Am Chem Soc. 2006 Mar 15;128(10):3166-8. PMID:16522096
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