LIGHT-DRIVEN ION TRANSPORT IN BACTERIAL RHODOPSINS

细菌视紫红质和盐视紫红质是最简单的离子泵， 普遍存在的七跨膜螺旋蛋白的原型。我们取得了 在过去几年中取得了不寻常的进展，现在建议测试和延长 由此产生的详细的一步一步的质子机制的假设， 运输它不仅包括蛋白质中的质子转移步骤， 也是单向性的热力学和结构原理， 离子移位的矢量性。这一全面的 假设现在开放的批判性检查，并在拟议的工作，我们 将特别注意许多未预测的突变表型 根据目前的版本。我们会继续采用现时的方法， 结合位点特异性诱变，时间分辨光谱， 高分辨率蛋白质X射线晶体学研究i）结构 质子释放步骤的问题，ii）可解离基团的pK如何 调节，iii）构象偶联的性质和原因，iv） 在不存在ASP-85和ASP-96的情况下质子传输的机制，和V） 从盐视紫红质和D85 T细菌视紫红质突变体中得到的教训， 既能传输氯离子，又能以与质子相反的方向传输。 本提案的目标是最终建立一个完整而明确的模型 这种离子泵的分子机制。

Bacteriorhodopsin and halorhodopsin are the simplest ion pumps, and prototypes of the ubiquitous seven-transmembrane-helical proteins. We have made unusual progress in the past years and now propose to test and extend the resulting detailed step-by-step hypothesis for the mechanism of proton transport. It includes not only the proton transfer steps in the protein but also the thermodynamic and structural rationales for the unidirectionality and the vectoriality of the ion translocation. All aspects of this comprehensive hypothesis are now open to critical examination, and in the proposed work we will pay particular attention to the numerous mutant phenotypes not predicted by its present version. We will continue to use our present approach of combining sitespecific mutagenesis, time-resolved spectroscopy, and high-resolution protein x-ray crystallography to investigate i) structural questions at the proton release step, ii) how the pKs of dissociable groups are modulated, iii) the nature and causes of conformational coupling, iv) the mechanism of proton transport in the absence of asp-85 and asp-96, and v) the lessons to be learned from halorhodopsin and the D85T bacteriorhodopsin mutant, that both transport chloride ions and in the opposite direction from protons. The goal of this proposal is to establish, finally, a full and explicit model for the molecular mechanism of this kind of ion pump.