PROTEIN DYNAMICS IN THE BACTERIORHODOPSIN PHOTOCYCLE - A NANOSECOND STEP-SCAN FTIR INVESTIGATION OF THE KL TO L TRANSITION

A time-resolved step-scan FTIR spectrometer with a time resolution of 20 ns was developed and used to investigate the KL to L transition in the photocycle of bacteriorhodopsin in the time range from -60 to 940 ns. Broadband FTIR absorbance difference spectra With a spectral range of 850-2050 cm(-1) and a spectral resolution of 4 cm(-1) have been ob tained. Our data show that there are two sets of BR photoproduct diffe rence bands exhibiting different kinetics. The intensity changes of ba nds attributed to structural changes of the carboxyl groups Asp-96 and Asp-115 as well as of bands assigned to C=C and C-C stretching modes of the chromophore show single exponential behavior with a time consta nt of about 400 ns, implying that these two processes are coupled. The time-dependent intensity changes of bands attributed to structural ch anges of the chromophore region near the Schiff base exhibit slower ki netics with a time constant of about 2 mu s We interpret our data in t erms of a process during the KL to L transition where structural chang es of the beta-ionone ring end of the chromophore and of Asp-115 occur faster than changes at the Schiff base region of the chromophore. Und er our physiologic al sample conditions, a perturbation of Asp-115 occ urs in the first 20 ns in contrast to results from hydrated films wher e this process is blocked or occurs more slowly. This fast protein res ponse indicates that there is direct coupling between the carboxylic a cid residues and the chromophore. Comparison of our data with low-temp erature and microsecond time-resolved infrared spectra of the L interm ediate in hydrated films of BR indicates that a different L structure is produced when the water activity is low.