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PDBsum entry 2o2b
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Luminescent protein
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PDB id
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2o2b
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References listed in PDB file
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Key reference
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Title
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Spectroscopic and structural study of proton and halide ion cooperative binding to gfp.
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Authors
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D.Arosio,
G.Garau,
F.Ricci,
L.Marchetti,
R.Bizzarri,
R.Nifosì,
F.Beltram.
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Ref.
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Biophys J, 2007,
93,
232-244.
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PubMed id
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Abstract
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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.
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