Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy

GFPs are upon excitation influenced by many different photophysical and pho tochemical processes effective over a very broad time scale. Much effort ha s been spent to investigate these processes. However, in the microsecond to millisecond time-range many processes still remain to be further character ized. This time-range can be conveniently covered by FCS, and is used here to study the photodynamical behaviour of wild-type (WT) and a F64L S65T mut ant (BioST) of GFP. In addition to intersystem crossing to the triplet stat e, additional photophysical processes are seen, showing identical fluctuati ons in fluorescence to those found for a reversible photo-induced isomeriza tion process, as well as fluctuations, not influenced by the electronic sta te of the chromophore unit. In the nanosecond time-range a contribution to the fluorescence correlation function is observed which can be attributed t o rotational diffusion, suggesting a convenient way to measure rotational d iffusion of proteins expressed with GFP on a microscopic scale. (C) 1999 El sevier Science B.V. All rights reserved.