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，也是无处不在的 七跨膜螺旋蛋白。我们在过去的几年里取得了不寻常的进展， 建议测试和扩展由此产生的详细的逐步假设的机制， 质子运输它不仅包括蛋白质中的质子转移步骤， 离子单向性和矢量性的热力学和结构原理 易位这一全面假设的所有方面现在都可以接受严格的审查， 在拟议的工作中，我们将特别注意许多突变表型， 根据目前的版本预测。我们将继续采用目前的方法，将现场- 特异性诱变、时间分辨光谱学和高分辨率蛋白质X射线晶体学 研究i）质子释放步骤的结构问题，ii）可解离的 iii）构象偶联的性质和原因，iv）构象偶联的机制， 在没有asp-85和asp-96的情况下的质子输运，以及v）从 盐视紫红质和D85T细菌视紫红质突变体，两者都运输氯离子，并在 与质子方向相反。本提案的目标是最终建立一个全面和明确的 建立了这种离子泵的分子机理模型。

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 site- specific mutagenesis, time-resolved spectroscopy, and high-resolution protein x-ray crystallography to investigate i) structural questions at the proton release step, ii) howthe pK's of dissociable groups are modulated, Hi)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.