Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 2. Unusual photophysical properties.

In the preceding accompanying paper [Shu, X., et al. (2007) Biochemistry 46, 12005-12013], the 1.5 A resolution crystal structure of green fluorescent protein (GFP) variant S65T/H148D is presented, and the possible consequences of an unusual short hydrogen bond (<or=2.4 A) between the carboxyl oxygen of Asp148 and the phenol oxygen of the chromophore are discussed. This work reports the femtosecond time-resolved emission of this variant at pH 5.6 by ultrafast fluorescence upconversion spectroscopy. Following excitation at 400 nm, green fluorescence is observed at 510 nm with a rise on a time scale that is faster than the 170 fs instrument response. Time-resolved emission spectra at 140 K also exhibit the immediate appearance of green fluorescence, and this extremely fast process is hardly affected by deuteration of exchangeable protons. These results appear to be dramatically different from those of wild-type GFP, in which the green fluorescence at 508 nm is produced on the picosecond time scale as a result of excited-state proton transfer from the state that is excited at 400 nm. The unique features observed in S65T/H148D and apparent ultrafast excited-state proton transfer are discussed in light of evidence for multiple states underlying the band at around 415 nm, as suggested by steady-state fluorescence spectra. The behavior of these different states may explain the novel photophysical properties observed for this GFP variant, including the ultrafast green fluorescence and the absence of completely matched decay in blue fluorescence. It is speculated that two different orientations of the Asp introduced at position 148, not distinguishable by chromatography, mass spectrometry, or X-ray crystallography, give rise to the two functionally distinct populations.