Femtosecond and picosecond fluorescence of native bacteriorhodopsin and a nonisomerizing analog.

The spectrally and temporally resolved fluorescence properties of native bacteriorhodopsin (bR) and bR reconstituted with a nonisomerizing analog of the retinal Schiff base (bR5.12) are examined. The first attempt to experimentally monitor the excited state relaxation processes in both type of pigments using ultrafast fluorescence spectroscopy is reported. The fluorescence is emitted from retinal molecules in an all-trans configuration. Substantial energy relaxation involves very fast intramolecular and intermolecular vibrational modes and these are shown to occur on a time scale faster than isomerization. The possible contribution of dielectric interaction between the retinal Schiff base and the protein environment for the excited state energy relaxation is discussed.

Spectral and kinetic fluorescence properties of native and nonisomerizing retinal in bacteriorhodopsin.

Steady-state and picosecond (ps) fluorescence studies of wild-type bacteriorhodopsin (wt-bR) and of a nonisomerizing analog locked in the all-trans configuration have been performed. Extending earlier work done by femtosecond absorption spectroscopy, we observe a strong similarity between both proteins in both fluorescence spectra and Stokes shift thus confirming the previous result that the fluorescent state I460 of the native bR proteins is in the all-trans configuration. Comparison of the spectra of fluorescence and stimulated emission of the locked pigments indicates the presence of an excited-state absorption situated around 750 nm. Upon increase of the excitation energy, the time-integrated fluorescence shows an interesting weak blue shift, which is identical for both pigments. Finally, we discuss the primary structural processes in retinal and in the protein that lead to the sub-100 fs formation of I460 and in particular to the considerable Stokes shift.