GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and 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 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-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 that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.
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.
We have used fluorescence spectroscopy techniques such as fluorescence correlation spectroscopy and fluorescence anisotropy decay on a wide time range, from nanoseconds to seconds, to investigate the unfolding kinetics induced by guanidinium chloride of GFPMut2 and its point mutation H148G, which has proved to be relevant for GFP photochemistry and photophysics. The mutation affects the unfolding kinetics of GFP leading to a much faster process at alkaline pH values, where protonation dynamics is negligible, that can be ascribed to a twofold role of His148, either as a proton shutter towards the chromophore and as a conformation stabiliser. For both mutants a soft region located near beta-strand 3 is found that starts to gain flexibility in the ns range at denaturant concentrations far lower than those required to turn off the chromophore fluorescence, as derived from the anisotropy decay of an extrinsic probe covalently bound to the proteins.
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