FTIR spectroscopy of bacteriorhodopsin microcrystals at Beamline 1.4

Bacteriorhodopsin (bR) is the sole protein component of the purple membrane of Halobacterium salinarium . The function of bR in vivo is to convert solar energy into a pH gradient across the cell membrane which the organism uses to drive ATP synthesis . Bacteriorhodopsin undergoes a light-induced cycle of physicochemical changes for for every proton it pumps out of the cell. The photocycle of bR has been wellcharacterized by both visible and IR spectroscopy. The major intermediates are identified as the K, L, M, N and O intermediates, and each has a distinct visible color and a distinct IR spectrum . The Schiff base that connects the side chain of Lys 216 to the retinal molecule buried within the core of the apoprotein 4 is deprotonated upon formation of the M intermediate, and reprotonated when the M intermediate decays. Since access to the Schiff base switches from the extracellular side of the membrane to the cytoplasmic side between these two proton transfer events, the M intermediate is of particular interest. High resolution x-ray diffraction experiments on microcrystals of bR have recently become possible, through the discovery by Landau and Rosenbusch that the solubilized protein can be crystallized from the bicontinuous lipid-water gel that is formed by mono-olein . Structural studies on intermediate states of the photocycle thus become a high priority, allowing the visualization of the structural changes that are responsible for converting light energy into a proton-motive force. Previously we collected high resolution x-ray diffraction data from crystals of wild type bR trapped in both the L and M states by illumination at low temperature, using Fourier Transform IR (FTIR) spectroscopy to confirm the identity of the photointermediate. This year we turned our attention this year to the F219L mutant of bR, which forms N, the intermediate following M, more readily than does the wild type protein. We have succeeded in characterizing two photointermediates of the bacteriorhodopsin mutant F219L by means of FTIR spectroscopy. At 213K an early phase of the M photostate is trapped, whereas at 173K the IR signature strongly suggests that the L photostate is trapped. Further studies of this mutant are planned, with an eye towards trapping the N intermediate.