1Z1P

Y66L variant of Enhanced Green Fluorescent Protein with 412-nm Absorbing Chromophore

Summary for 1Z1P

• Entry DOI: 10.2210/pdb1z1p/pdb
• Related: 1S6Z
• Descriptor: green fluorescent protein (2 entities in total)
• Functional Keywords: gfp, beta barrel, uv/vis absorbing yellow chromophore, luminescent protein
• Biological source: Aequorea victoria
• Total number of polymer chains: 1
• Total formula weight: 26879.37

Authors

Rosenow, M.A.,Patel, H.N.,Wachter, R.M. (deposition date: 2005-03-04, release date: 2005-06-21, Last modification date: 2024-11-13)

Primary citation

Rosenow, M.A.,Patel, H.N.,Wachter, R.M.
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.
Biochemistry, 44:8303-8311, 2005

PubMed 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.

PubMed: 15938620
DOI: 10.1021/bi0503798

Experimental method

X-RAY DIFFRACTION (2 Å)