1yj2
From Proteopedia
(New page: 200px<br /><applet load="1yj2" size="450" color="white" frame="true" align="right" spinBox="true" caption="1yj2, resolution 1.50Å" /> '''Cyclized, non-dehydr...) |
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| - | [[Image:1yj2.gif|left|200px]]<br /><applet load="1yj2" size=" | + | [[Image:1yj2.gif|left|200px]]<br /><applet load="1yj2" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1yj2, resolution 1.50Å" /> | caption="1yj2, resolution 1.50Å" /> | ||
'''Cyclized, non-dehydrated post-translational product for S65A Y66S H148G GFP variant'''<br /> | '''Cyclized, non-dehydrated post-translational product for S65A Y66S H148G GFP variant'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The Aequorea victoria green fluorescent protein (GFP) undergoes a | + | The Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable post-translational modification to create a chromophore out of its component amino acids S65, Y66, and G67. Here, we describe mutational experiments in GFP designed to convert this chromophore into a 4-methylidene-imidazole-5-one (MIO) moiety similar to the post-translational active-site electrophile of histidine ammonia lyase (HAL). Crystallographic structures of GFP variant S65A Y66S (GFPhal) and of four additional related site-directed mutants reveal an aromatic MIO moiety and mechanistic details of GFP chromophore formation and MIO biosynthesis. Specifically, the GFP scaffold promotes backbone cyclization by (1) favoring nucleophilic attack by close proximity alignment of the G67 amide lone pair with the pi orbital of the residue 65 carbonyl and (2) removing enthalpic barriers by eliminating inhibitory main-chain hydrogen bonds in the precursor state. GFP R96 appears to induce structural rearrangements important in aligning the molecular orbitals for ring cyclization, favor G67 nitrogen deprotonation through electrostatic interactions with the Y66 carbonyl, and stabilize the reduced enolate intermediate. Our structures and analysis also highlight negative design features of the wild-type GFP architecture, which favor chromophore formation by destabilizing alternative conformations of the chromophore tripeptide. By providing a molecular basis for understanding and controlling the driving force and protein chemistry of chromophore creation, this research has implications for expansion of the genetic code through engineering of modified amino acids. |
==About this Structure== | ==About this Structure== | ||
| - | 1YJ2 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 and EDO as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http:// | + | 1YJ2 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> and <scene name='pdbligand=EDO:'>EDO</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1YJ2 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: Kassmann, C | + | [[Category: Kassmann, C J.]] |
| - | [[Category: Tainer, J | + | [[Category: Tainer, J A.]] |
[[Category: EDO]] | [[Category: EDO]] | ||
[[Category: MG]] | [[Category: MG]] | ||
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[[Category: mio]] | [[Category: mio]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:05:48 2008'' |
Revision as of 14:05, 21 February 2008
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Cyclized, non-dehydrated post-translational product for S65A Y66S H148G GFP variant
Overview
The Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable post-translational modification to create a chromophore out of its component amino acids S65, Y66, and G67. Here, we describe mutational experiments in GFP designed to convert this chromophore into a 4-methylidene-imidazole-5-one (MIO) moiety similar to the post-translational active-site electrophile of histidine ammonia lyase (HAL). Crystallographic structures of GFP variant S65A Y66S (GFPhal) and of four additional related site-directed mutants reveal an aromatic MIO moiety and mechanistic details of GFP chromophore formation and MIO biosynthesis. Specifically, the GFP scaffold promotes backbone cyclization by (1) favoring nucleophilic attack by close proximity alignment of the G67 amide lone pair with the pi orbital of the residue 65 carbonyl and (2) removing enthalpic barriers by eliminating inhibitory main-chain hydrogen bonds in the precursor state. GFP R96 appears to induce structural rearrangements important in aligning the molecular orbitals for ring cyclization, favor G67 nitrogen deprotonation through electrostatic interactions with the Y66 carbonyl, and stabilize the reduced enolate intermediate. Our structures and analysis also highlight negative design features of the wild-type GFP architecture, which favor chromophore formation by destabilizing alternative conformations of the chromophore tripeptide. By providing a molecular basis for understanding and controlling the driving force and protein chemistry of chromophore creation, this research has implications for expansion of the genetic code through engineering of modified amino acids.
About this Structure
1YJ2 is a Single protein structure of sequence from Aequorea victoria with and as ligands. Full crystallographic information is available from OCA.
Reference
Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry., Barondeau DP, Kassmann CJ, Tainer JA, Getzoff ED, Biochemistry. 2005 Feb 15;44(6):1960-70. PMID:15697221
Page seeded by OCA on Thu Feb 21 16:05:48 2008
Categories: Aequorea victoria | Single protein | Barondeau, D P. | Getzoff, E D. | Kassmann, C J. | Tainer, J A. | EDO | MG | Ammonia | Biosynthesis | Chromophore | Electrophile | Hal | Histidine | Lyase | Mio
