2o2b

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Spectroscopic and Structural Study of the Heterotropic Linkage between Halide and Proton Ion Binding to Gfp Proteins: E2(GFP)-I Complex

Overview

This study reports the influence of halogens on fluorescence properties of, the Aequorea victoria Green Fluorescent Protein variant S65T/T203Y, (E2GFP). Halide binding forms a specific non-fluorescent complex, generating a substantial drop of the fluorescence via static quenching., Spectroscopic analysis under different solution conditions reveals high, halogen affinity, which is strongly dependent on the pH. This evidences, the presence in E(2)GFP of interacting binding sites for halide ions and, for protons. Thermodynamic link and cooperative interaction are assessed, demonstrating that binding of one halide ion is associated with the, binding of one proton in a cooperative fashion with the formation, in the, pH range 4.5-10, of a single fully protonated E(2)GFP.halogen complex. In, order to resolve the structural determinants of E(2)GFP sensitivity to, halogens high-resolution crystallographic structures were obtained for the, halide-free and I(-), Br(-) and Cl(-) bound E(2)GFP. Remarkably the first, high-resolution (1.4 A) crystallographic structure of a chloride-bound GFP, is reported. The chloride ion occupies a specific and unique binding, pocket in direct contact (3.4 A) with the chromophore imidazolidinone, aromatic ring. Unanticipated flexibility, strongly modulated by halide ion, interactions, is observed in the region surrounding the chromophore., Furthermore molecular dynamics simulations identified E222 residue (along, with the chromophore Y66 residue) being in the protonated state when, E(2)GFP.halogen complex is formed. The impact of these results on, high-sensitivity biosensor design will be discussed.

About this Structure

2O2B is a Single protein structure of sequence from Aequorea victoria with as ligand. Full crystallographic information is available from OCA.

Reference

Spectroscopic and Structural study of Proton and Halide ion cooperative binding to GFP., Arosio DP, Garau G, Ricci F, Marchetti L, Bizzarri R, Nifosi R, Beltram F, Biophys J. 2007 Apr 13;. PMID:17434942

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