LIGHT-DRIVEN ION TRANSPORT IN BACTERIAL RHODOPSINS

细菌视紫红质和卤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。我们已经做出了 在过去的几年中取得了不同寻常的进展，现在建议测试和扩展 由此产生的质子机制的详细逐步假说 运输。它不仅包括蛋白质中的质子转移步骤，还包括 此外，单方向性和单向性的热力学和结构原理 离子移位的矢向性。这一全面的 假说现在可以进行批判性检验，在拟议的工作中，我们 将特别关注众多未被预测的突变表型 根据目前的版本。我们将继续使用我们目前的方法 结合定点突变、时间分辨光谱和 高分辨蛋白质X-射线结晶学研究I)结构 质子释放步骤中的问题，II)可解离基团的PKS是如何 三)构象偶联的性质和原因，四)构象偶联 在没有asp-85和asp-96的情况下的质子输运机制，以及v) 从嗜盐紫红质和D85T细菌视紫红质突变体中吸取的教训， 这两种物质都能传输氯离子，而且与质子的传输方向相反。 这项提议的目标是最终建立一个完整和明确的模型 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.