Four Dimensional Structural Analysis of Biological Ion Pumps

细菌视紫红质是一种存在于盐细菌细胞膜上的光驱动质子泵，是一种具有良好特性的生物离子泵。基于四维X射线晶体学分析的结果，首席研究员和同事提出了一个新的假设，即细菌视紫红质实际上是水和质子的反向转运体。为了支持这一假设，我们研究了时间分辨光谱以及暴露于加压惰性气体的细菌视紫红质的X射线晶体学分析。然后发现，当λ或Kr原子特异性地结合到细菌视紫红质的胞质侧的疏水腔中时，M中间体的衰变显著加速。我们还进行了古紫质-2的X射线晶体学分析，这是一种在Halorubrum中发现的光驱动质子泵，并以2.1埃的分辨率确定了其三维结构。结果表明，古视紫红质-2的晶体是由堆叠的膜，在每个膜中的三聚体复合物被安排在蜂窝晶格和第二生色团，细菌红蛋白，介导的三聚体内相邻的蛋白质之间的相互作用。我们成功地得到了变形菌视紫红质的三维晶体，这是一种来自变形菌的光驱动质子泵。我们已经建立了制备大量的盐视紫红质，光驱动的氯离子泵，经过反复循环的紫外诱变。我们得到了一个有序的三维晶体的鱿鱼视紫红质，并确定其结构在2.8埃的分辨率。

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.