The goal of this Project is to understand the exact molecular mechanism by which bacteriorhodopsin uses light energy to pump protons across the cell membrane. It is already established that bacteriorhodopsin cycles between two distinct protein conformations in which the (proton-binding) """"""""active site"""""""" is first exposed to the extracellular side of the membrane and then later to the cytoplasmic side of the membrane. What is still lacking are atomic-resolution data on the way in which photochemical changes in the retinal ligand lead to changes in protein conformation, and the processes by which these changes in protein conformation ultimately lead to restoration of the original, all trans, protonated Schiff base configuration of the retinal group. Arguable even whether the actual mass- species which is transported to the hydroxyl ion, which would be pumped into the cytoplasm. Electron diffraction difference maps will be used to observe high-resolution changes in protein structure which occur at different intermediate stages in the ion-pumping photocycle. Low- temperature trapping methods and the use of well-characterized mutant proteins will be used to prepare samples in spectroscopically will defined intermediate states. Included in this work will be the single-site mutant E204Q, which pumps chloride ions rather than protons (hydroxyl ions?). A comparison of the structural changes observed in this mutant and those observed in wild-type bacteriorhodopsin should held to understand whether wild-type bR is actually a hydroxyl-ion pump. In a major new direction for our work, the recently reported method of growing three-dimensional microcystals of bacteriorhodopsin will be exploited in order to use x-ray diffraction data as well as the electron diffraction data that have been the method of choice up until now. In both cases, three-dimensional difference Fourier maps will be used to rebuild the existing atomic resolution model of the resting state of bacteriorhodopsin in the regions where significant structural changes has occurred, and diffraction data is higher than o.3 nm resolution will be used to refine the rebuilt mode.
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