DYNAMICS OF FLUORESCENCE FLUCTUATIONS IN GREEN FLUORESCENT PROTEIN OBSERVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

We have investigated the pH dependence of the dynamics of conformation al fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluo rescence correlation spectroscopy (FCS), FCS utilizes time-resolved me asurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all proc esses that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pK(a) = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. P rotonation of the hydroxyl group of Tyr-66, which is part of the chrom ophore, induces these changes. When FCS is used the fluctuations in th e protonation state of the chromophore are time resolved, The autocorr elation function of fluorescence emission shows contributions from two chemical relaxation precesses as well as diffusional concentration fl uctuations. The time constant of the fast, pH-dependent chemical proce ss decreases with pH from 300 mu s at pH 7 to 45 mu s at pH 5, while t he time-average fraction of molecules in a nonfluorescent state increa ses to 80% in the same range. A second, pH-independent, process with a time constant of 340 mu s and an associated fraction of 13% nonfluore scent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rea rrangements. The FCS data pro,ide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in su bfemtoliter volumes with nanomolar concentrations of EGFP.