Talk at the International Sumposium on GFP, Oct. 18-22 1997 in New Brunswick NJ Fluorescence correlation spectroscopy (Magde et al. 1972, Phys. Rev. Lett. 29:705) is uniquely suited for determination of the rate constants of molecular dynamics and chemical kinetics of spontaneous processes. Statistical physics dictates that the fluorescence obtained from the small probe volume employed in FCS fluctuates about a mean value, due to individual molecules diffusing in and out of the volume and/or due to inter-conversion of such molecules to and from a non-fluorescent form. The autocorrelation function therefore contains information about chemical reaction kinetics, coefficients of diffusion and the equilibrium chemical concentrations. GFP fluorescence is highly dependent on the protonation state and external conditions (Ward et al. 1982, Photochem. Photobiol. 35:803-808). Using one-photon excitation (488nm) with confocal fluorescence detection and alternatively two-photon excitation (910nm), we have identified a spontaneous modulation of the fluorescence with a charact eristic time constant around 200 (s. The amplitude of the modulation is pH dependent; with decreasing pH an increasing fraction of molecules is found in a nonfluorescent state (in agreement with bulk measurements). The kinetics are temperature dependent yielding an activation energy of about 10 kcal/mol and are slowed down in D2O by a factor of about 1.5. From these results we conclude that the observed kinetics represent thermal interconversion between two states of the fluorophore (Brejc et al. 1997, Proc. Acad. Natl. Sci. USA 94:2306-2311): protonated (?abs~400nm, not well-excited by 488nm) and unprotonated (?abs~490nm). This process is distinguished from that recently reported (Dickson et al., Nature 388:355-358) at much slower time-scales (~1s).
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