title: Mutagenic stabilization of the photocycle intermediate of green fluorescent protein (GFP).
abstract: The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited-state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild-type(wt)-GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.
authors: Genzentrum der Ludwig-Maximilians-Universität, Feodor-Lynen Strasse 25, 81377 Münich, Germany.
authors: Jens
authors: J
authors: Wiehler
authors: Gregor
authors: G
authors: Jung
authors: Christian
authors: C
authors: Seebacher
authors: Andreas
authors: A
authors: Zumbusch
authors: Boris
authors: B
authors: Steipe
mesh_terms: D000042:Absorption; D000818:Animals; D019281:Dimerization; D049452:Green Fluorescent Proteins; D037402:Hydrozoa / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins