LIGHT-DRIVEN ION TRANSPORT IN BACTERIAL RHODOPSINS

膜离子泵尚未解决的基本问题可以研究到 细菌视紫红质和嗜盐视紫红质的最大优势， 光驱生电离子泵在盐生盐杆菌中表现最好 表征了活性转运蛋白。使用我们在中开发的模型 在过去的几年里，关于光反应序列和 在这些蛋白质中的离子转运，我们将描述特定的机制 输运循环的步骤和生色团之间的耦合 反应和产生的离子梯度。通过比较两个非常相似的 传输我们期望获得的不同离子的传输系统 机械的和概念性的见解，并期望这些将是相关的 到一般的离子泵。将使用各种方法来 评估特定氨基酸残基在结构和结构中的作用 这些小视网膜蛋白的功能，最重要的是通过位点特异性 诱变。细菌视紫红质和巴氏视紫红质 将生成多个残留物变化，使用最近开发的 嗜盐杆菌穿梭载体及转化系统 将已克隆的bop和hop基因导入并表达。这些与之对应的 表达的蛋白质与先前表达的蛋白质有不同的表型 在大肠杆菌中。突变方法将得到替代的补充。 与视网膜类似物的视网膜与改变后的结合相适应 口袋里。我们将用时间分辨光学技术分析改变后的蛋白质 低温多通道和单波长光谱分析 光谱学和细胞膜囊泡的运输研究。穿过 合作FTIR和共振拉曼光谱，光电和 电光测量、视网膜类似物和x射线衍射将 如有需要，请随时与我们联系。

The fundamental unsolved questions of membrane ion pumps can be studied to greatest advantage with bacteriorhodopsin and halorhodopsin, the light-driven electrogenic ion pumps in, Halobacterium halobium, the best characterized active transport proteins. Using the models we developed in the last few years for the photoreaction sequences and the energetics of ion transport in these proteins, we will describe the mechanism of specific steps of the transport cycles and the coupling between the chromophore reactions and the ion gradients created. By comparing two closely similar transport systems which transport different ions we expect to gain mechanistic and conceptual insights, and expect that these will be relevant to ionic pumps in general. A variety of approaches will be used to evaluate the role of specific amino acid residues in the structure and function of these small retinal proteins, most importantly by site-specific mutagenesis. Bacteriorhodopsins and balorhodopsins with single and multiple residue changes will be generated, using a recently developed shuttle vector and transformation system for Halobacterium halobium to introduce and express the cloned bop and hop genes. These homologously expressed proteins have different phenotypes from those expressed earlier in E. coli. The mutagenesis approach will be complemented by replacement of the retinal with retinal analogues with altered fit into the binding pocket. We will analyze the altered proteins with time-resolved optical multichannel and single wavelength spectroscopy, low temperature spectroscopy, and transport studies in cell envelope vesicles. Through collaborations FTIR and resonance Raman spectroscopy, photoelectric and electro-optical measurements, retinal analogues, and x-ray diffraction will be available to us when needed.