Four Dimensional Structural Analysis of Biological Ion Pumps

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Bacteriorhodopsin, a light-driven proton pump found in the cell membrane of halobacteria, is one of well characterized biological ion pumps. Based on the results of four-dimensional X-ray crystallographic analyses, the principal investigator and colleague have proposed a new hypothesis that bacteriorhodopsin actually functions as an antiporter of water and proton. To get supports for this hypothesis, we investigated time-resolved spectroscopy as well as X-ray crystallographic analysis of bacteriorhodopin exposed to pressurized noble gases. It was then found that when Xe or Kr atom binds specificically to a hydrophobic cavity in the cytoplasmic side of bacteriorhodopsin, the decay of the M intermediate is accelerated significantly. We also performed X-ray crystallographic analysis of archaearhodopsin-2, a light-driven proton pump found in Halorubrum, and determined its three-dimensional structure at 2.1 angstrom resolution. It was shown that the crystal of archaerhodopsin-2 is made up of stacked membranes, in each of which the trimeric complexes are arranged on a honeycomb lattice and that the second chromophore, bacterioruberin, mediates interactions between neighboring proteins within the trimer. We succeeded in getting a three-dimensional crystal of proteorhodopsin, a light-driven proton pump from a proteobacterioum. We have established to prepare a large amount of halorhodopsin, a light-driven chloride pump, after a repeated cycle of UV mutagenesis. We obtained a well-ordered three-dimensional crystal of squid rhodopsin and determined its structure at 2.8 angstrom resolution.