TITLE: Unraveling the Role of Arginine 82 in the Bacteriorhodopsin Photocycle

SPEAKER: Dr. M. Shane Hutson,

TIME: Thursday Apr. 19, 2001 at 4 PM

PLACE: George P. Williams, Jr. Lecture Hall, (Olin 101)

The bacteriorhodopsin (bR) protein functions as a light-driven proton pump; i.e. it converts solar energy into an electrochemical proton gradient across the membranes of halophilic archaebacteria. The molecular events responsible for energy conversion have been the subject of intense research for several decades. Arginine 82 has long been recognized as an important residue of bR due to its absolute conservation in the archaeal rhodopsin protein family and the effects of R82 mutations on the photocycle. However, the nature of R82's interactions with other residues of the protein has remained difficult to decipher. Recent NMR studies showed that R82 enters a highly asymmetric environment during the M state trapped at cryogenic temperatures [Petkova et al, Biochemistry 38: 1562-72, 1999]. We have measured the time-resolved FT-IR spectra of bR intermediates in the wild-type and R82A mutant proteins on the microsecond time scale. The results indicate that there is a significant perturbation of R82 associated with proton release in the bR photocycle. Although the pKa of arginine is normally >13 in aqueous solution, the nature of the spectral changes suggests that R82 deprotonates in the bR photocycle at pH 9.5. Recent work designed to extend time-resolved, broadband infrared measurements into the picosecond time-regime with a novel two-color beamline under construction at the Duke Free Electron Laser Lab will also be presented. This beamline offers the capability to excite systems with tunable ultraviolet radiation and then probe the relaxation processes throughout the infrared with time resolution below 100 ps. The beamline opens the doors to the study of photochemical and photobiological systems not previo usly accessible to time-resolved, broadband infrared spectroscopy.