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| - | [[Image:1s6z.gif|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_1s6z| PDB=1s6z | SCENE= }} | | {{STRUCTURE_1s6z| PDB=1s6z | SCENE= }} |
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| - | '''Enhanced Green Fluorescent Protein Containing the Y66L Substitution'''
| + | ===Enhanced Green Fluorescent Protein Containing the Y66L Substitution=== |
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| - | ==Overview==
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| - | The crystal structure of a colorless variant of green fluorescent protein (GFP) containing the Y66L substitution has been determined to 1.5 A. Crystallographic evidence is presented for the formation of a trapped intermediate on the pathway of chromophore maturation, where the peptide backbone of residues 65-67 has condensed to form a five-membered heterocyclic ring. The hydroxyl leaving group remains attached to the ring as confirmed by high-resolution electrospray mass spectrometry. The alpha-carbon of residue 66 exhibits trigonal planar geometry, consistent with ring oxidation by molecular oxygen. Side chain positions of surrounding residues are not perturbed, in contrast to structural results obtained for the GFPsol-S65G/Y66G variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116]. The data are in accord with a reaction pathway in which dehydration is the last of three chemical steps in GFP chromophore formation. A novel mechanism for chromophore biosynthesis is proposed: when the protein folds, the backbone condenses to form a cyclopentyl tetrahedral intermediate. In the second step, the ring is oxidized by molecular oxygen. In the third and final step, elimination of the hydroxyl leaving group as water is coupled to a proton transfer reaction that may proceed via hydrogen-bonded solvent molecules. Replacement of the aromatic Tyr66 with an aliphatic residue appears to have a profound effect on the efficiency of ring dehydration. The proposed mechanism has important implications for understanding the factors that limit the maturation rate of GFP. | + | The line below this paragraph, {{ABSTRACT_PUBMED_15078092}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 15078092 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_15078092}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Gfp-like protein]] | | [[Category: Gfp-like protein]] |
| | [[Category: Trapped intermediate]] | | [[Category: Trapped intermediate]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 08:22:35 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 17:16:02 2008'' |
Revision as of 14:16, 28 July 2008
Template:STRUCTURE 1s6z
Enhanced Green Fluorescent Protein Containing the Y66L Substitution
Template:ABSTRACT PUBMED 15078092
About this Structure
1S6Z is a Single protein structure of sequence from Synthetic construct. Full crystallographic information is available from OCA.
Reference
The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization-oxidation-dehydration mechanism for chromophore maturation., Rosenow MA, Huffman HA, Phail ME, Wachter RM, Biochemistry. 2004 Apr 20;43(15):4464-72. PMID:15078092
Page seeded by OCA on Mon Jul 28 17:16:02 2008
