Oxidative Chemistry in the GFP Active Site Leads to Covalent Cross-Linking of a Modified Leucine Side Chain with a Histidine Imidazole:  Implications for the Mechanism of Chromophore Formation^,

Abstract

The mechanism of chromophore biosynthesis in green fluorescent protein (GFP) is triggered by a spontaneous main chain cyclization reaction of residues 65-67. Here, we demonstrate that the initially colorless Y66L variant, designed to trap chromophore precursor states, is oxidatively modified to generate yellow chromophores that absorb at 412 and 374 nm. High- and low-pH crystal structures determined to 2.0 and 1.5 A resolution, respectively, are consistent with pi-orbital conjugation of a planar Leu66-derived adduct with the imidazolinone ring, which is approximately 90 and 100% dehydrated, respectively. Time-, base-, and oxygen-dependent optical properties suggest that the yellow chromophores are generated from a 338 nm-absorbing intermediate, interpreted to be the Y66L analogue of the wild-type GFP chromophore. Generation of this species is catalyzed by a general base such as formate, and proceeds via a cyclization-oxidation-dehydration mechanism. The data suggest that a hydration-dehydration equilibrium exists in the cyclic form of the peptide, and that dehydration is favored upon extensive conjugation with the modified side chain. We conclude that the mechanism of GFP chromophore biosynthesis is not driven by the aromatic character of residue 66. In the low-pH X-ray structure, a highly unusual cross-link is observed between His148 and the oxidized Leu66 side chain, suggesting a conjugate addition reaction of the imidazole nitrogen to the highly electrophilic diene group of the yellow chromophore. The reactivity described here further expands the chemical diversity observed in the active site of GFP-like proteins, and may allow for covalent attachment of functional groups to the protein scaffold for catalytic purposes.

Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus

“…GFP folds into an 11-stranded β-barrel with a single helical segment threaded through the center of the barrel, and capped by several loops at either end of the barrel. A sharp turn places strain on three residues within the barrel-encapsulated stretch, which consequently drives cyclization via a dehydration/oxidation mechanism that requires only molecular oxygen678. The cyclized chromophore remains covalently attached to the polypeptide chain, and therefore, every cell expressing the GFP gene acquires fluorescence.…”

Kinetic Isotope Effect Studies on the de Novo Rate of Chromophore Formation in Fast- and Slow-Maturing GFP Variants

“…These compounds were able to diffuse into the protein's interior and accept a proton under physiological conditions (16, 17). Redox chemistry did not play a role in re-hybridization from sp 3 to sp 2 , rendering a radical mechanism for C β 66 oxidation unlikely (14). Here, we present further evidence that the chemistry observed in Y66L is analogous to the normal maturation mechanism of intact GFPs.…”

Mechanistic aspects of GFP chromophore biogenesis

“…We also found that the yellow species is generated from an intermediate that absorbs at 338 nm, and that formation of the 338-nm species is independent of oxygen. The X-ray structure of yellow Y66L indicated nearly complete elimination of water from the heterocycle (21). The electron density of the leucine side chain appeared to be in a near-planar conformation, most consistent with π-orbital conjugation of the side chain atoms with the heterocycle.…”

“…Procedures were carried out as described in reference (21). Colorless Y66L was converted to a UV/Vis absorbing form with yellow appearance by incubation in the presence of 2.0 M sodium formate.…”

“…Procedures were carried out as described in references (17,21). EGFP-Y66L was expressed in soluble form and crystallized via the hanging-drop vapor diffusion method with polyethylene glycol as precipitant.…”