2O29

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

Summary for 2O29

• Entry DOI: 10.2210/pdb2o29/pdb
• Related: 2H6V 2O24 2O2B
• Descriptor: Green fluorescent protein, BROMIDE ION (3 entities in total)
• Functional Keywords: luminescence, green fluorescent protein, gfp, e2, bioluminescence, photoactive protein, fluorescent chloride, bromide, iodine, halogen, luminescent protein
• Biological source: Aequorea victoria
• Total number of polymer chains: 1
• Total formula weight: 27837.63

Authors

Garau, G. (deposition date: 2006-11-29, release date: 2007-05-15, Last modification date: 2024-11-13)

Primary citation

Arosio, D.,Garau, G.,Ricci, F.,Marchetti, L.,Bizzarri, R.,Beltram, F.
Spectroscopic and Structural Study of Proton and Halide Ion Cooperative Binding to GFP.
Biophys.J., 93:232-244, 2007

PubMed Abstract: This study reports the influence of halogens on fluorescence properties of the Aequorea victoria Green Fluorescent Protein variant S65T/T203Y (E(2)GFP). Halide binding forms a specific nonfluorescent 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. 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.

PubMed: 17434942
DOI: 10.1529/biophysj.106.102319

Experimental method

X-RAY DIFFRACTION (1.8 Å)