New probes for kinetic intracellular measurements in the millisecond range are desirable to monitor protein biochemical dynamics essential for catalysis, allosteric regulation, and signaling. Good candidates to this aim are the photoswitchable mutants of the green fluorescent protein, whose anionic fluorescence, primed by blue light, is markedly enhanced under an additional excitation at a shorter wavelength and relaxes within a few milliseconds. The aim of this report is to study how the brightness enhancement kinetics depends on the physical-chemical and spectroscopic parameters and to provide proof-of-concept experiments for the use of the fluorescence enhancement in conditions in which the protein diffusion is hindered and thereby photobleaching can be a limiting critical issue. Future, direct applications of photochromic mutants for modulated excitation imaging would in fact require such a detailed knowledge. We present here an extensive study of the photoswitching mechanism of the E222Q mutant of GFPMut2 (Mut2Q), pumped by visible 488 nm light and probed at 400-420 nm, as a function of pH, viscosity, temperature, and light intensity. In solution, two characteristic photoswitching times are found by means of modulated double beam fluorescence correlation spectroscopy in the 1-30 ms range, depending on the solution pH. The photoswitching kinetics is solved in terms of the eigenvalues and the eigenvectors of a specific energy diagram and used directly to fit the data, suggesting that the observed photoswitching amplitudes and kinetics are related to a single three-level transition loop. Finally, we give in vitro examples of the use of modulated excitation microscopy, based on fluorescence enhancement amplitude and kinetics detection, on Mut2Q protein samples immobilized in acrylamide gels.

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