GFP (Green Fluorescent Proteins) mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Since serine 65, threonine 203, glutamate 222 and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we focus on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 ms pH dependent component and a 100-500 ms laser power dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a three-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited state process probably related to conformational rearrangements of the chromophore after the photo-excitation, more than to the chromophore excited state proton transfer.

Fondazione Cariplo (2005-1079).

University of Parma: S. Bettati, A. Mozzarelli.

Maddalena Collini.

• Daglio;Banterle; D’Alfonso; Collini; Chirico. Diffusion_Photodynamics Coupling in Fluorescence Correlation Spectroscopy Studies of Photoswitchable Green Fluorescent Proteins: An Analytical and Simulative Study. J. Phys. Chem. B, 2011, 115: 10311-10321.
• Quercioli, V, Bosisio, C., Daglio, S.C., Rocca, F., D’Alfonso, F., Collini, M., Baldini, G. and Chirico, G., Bettati, S. Raboni, S. , Campanini, B. Photo-Induced Millisecond Switching Kinetics In the Gfpmut2 E222q Mutant, J. Phys. Chem. B. (2010) 114(13): 4664-4677.
• Bosisio C., V. Quercioli, M. Collini, L. D’Alfonso, G. Baldini, S. Bettati, B. Campanini, S. Raboni, and G. Chirico, J.Phys.Chem.B. Protonation and Conformational Dynamics of GFP Mutants by Two-Photon Excitation Fluorescence Correlation Spectroscopy. 2008; 112(29); 8806-8814.