1S6Z

Enhanced Green Fluorescent Protein Containing the Y66L Substitution

Summary for 1S6Z

• Entry DOI: 10.2210/pdb1s6z/pdb
• Related: 1EMG 1QYF 1RM9 1RMO 1RMP
• Descriptor: green fluorescent protein, CHLORIDE ION (3 entities in total)
• Functional Keywords: chromophore, eleven-stranded beta barrel, trapped intermediate, backbone cyclization, gfp-like protein, luminescent protein
• Biological source: synthetic construct
• Total number of polymer chains: 1
• Total formula weight: 26914.82

Authors

Rosenow, M.A.,Huffman, H.A.,Phail, M.E.,Wachter, R.M. (deposition date: 2004-01-28, release date: 2004-05-04, Last modification date: 2024-10-30)

Primary citation

Rosenow, M.A.,Huffman, H.A.,Phail, M.E.,Wachter, R.M.
The Crystal Structure of the Y66L Variant of Green Fluorescent Protein Supports a Cyclization-Oxidation-Dehydration Mechanism for Chromophore Maturation(,).
Biochemistry, 43:4464-4472, 2004

PubMed Abstract: 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.

PubMed: 15078092
DOI: 10.1021/bi0361315

Experimental method

X-RAY DIFFRACTION (1.5 Å)