Four Dimensional Structural Analysis of Biological Ion Pumps

细菌视紫红质是一种存在于嗜盐细菌细胞膜上的光驱动质子泵，是一种特性良好的生物离子泵。基于四维X射线晶体分析的结果，主要研究人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。为了支持这一假说，我们研究了暴露在加压惰性气体中的细菌视紫红质的时间分辨光谱学和X射线结晶学分析。结果发现，当Xe或Kr原子与细菌视紫红质胞质侧的疏水空腔结合时，M中间体的衰变速度显著加快。我们还对古视紫质-2进行了X射线结晶学分析，并在2.1埃分辨率下确定了它的三维结构。结果表明，古紫质-2的晶体是由堆积的膜组成的，其中每个三聚体都以蜂窝状的晶格排列，而第二个发色团细菌红绿素在三聚体中介导了相邻蛋白质之间的相互作用。我们成功地获得了蛋白视紫红质的三维晶体，这是一种来自蛋白细菌的光驱动质子泵。我们已经建立了在经过反复的紫外线诱变循环后，制备大量的卤视紫质，一种光驱动的氯离子泵。我们得到了有序的鱿鱼视紫红质三维晶体，并测定了其2.8埃分辨率的结构。

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.