1s6z
From Proteopedia
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Enhanced Green Fluorescent Protein Containing the Y66L Substitution
Overview
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.
About this Structure
1S6Z is a Single protein structure of sequence from Synthetic construct with CL as ligand. 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 Sat Nov 24 23:59:56 2007
