The Crystal Structure of the Y66L Variant of Green Fluorescent Protein Supports a Cyclization−Oxidation−Dehydration Mechanism for Chromophore Maturation^,

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.

Structural Evidence for a Dehydrated Intermediate in Green Fluorescent Protein Chromophore Biosynthesis

“…4c) trapped in the crystal of the colorless EGFP variant. This variant has the chromophore-forming sequence Thr-65-Leu-66 -Gly-67 with an unusual non-aromatic Y66L substitution (31). The observed form reveals the hydroxylated imidazolone and partially oxidized Leu-66 C ␣ -C ␤ bond with sp 2 and sp 3 hybridization of the respective atoms.…”

“…Several crystal structures trapping various intermediate states have been reported, leading to a number of proposed reaction schemes of chromophore biosynthesis (21,31,32). One of the plausible pathways of chromophore maturation (Fig.…”

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

“…A similar reversible hydration reaction was postulated, although controversially discussed, to occur during the chromophore formation of GFP [27][28][29] . This might suggest that the light-induced reversible switching of Dreiklang is based on a molecular reaction that is possibly occurring during chromophore maturation of some fluorescent proteins.…”

A conserved interaction with the chromophore of fluorescent proteins

“…6). [46][47][48][49][50][51] A number of crystal structures are known for the conformation of the precyclized state, and these structures are of two distinct types. In one type of precyclized structure, an n!p* interaction exists between the main-chain carbonyl group of residue 62 and the chromophore [d ¼ 3.0 Å , y ¼ 97 ; Fig.…”

“…7(B)] could make the electrophilic and nucleophilic centers more reactive simultaneously. Two other on-pathway intermediates, namely the cyclized 47 and dehydrated structures, 51 have considerable n!p* interaction between the donor carbonyl group of residue 62 and the acceptor carbonyl group of the imidazolidinone ring [Figs. 7(C,D), Supporting Information Table S2].…”