TIME-RESOLVED VIBRATIONAL SPECTROSCOPY OF HALORHODOPSIN

氯离子跨细胞膜运输的生理意义 最近的一项研究表明，基因缺陷 在参与氯离子转运的单个跨膜蛋白中 导致囊性纤维化。然而，尽管在基因方面取得了这样的进展 膜转运氯化物的详细机制 人们对蛋白质的了解很少，这在很大程度上是因为缺乏蛋白质 关于阴离子间相互作用动力学的基本信息 以及它们在运输蛋白上的结合位置。我们会申请 时间分辨傅里叶变换红外光谱分析 生物膜对活性氯离子迁移的研究。这个 要研究的模型系统是卤视紫质(HR)，一种光驱动的阴离子 在几个物种的细胞膜中发现的转运蛋白 古细菌。HR和HR的TR-FTIR振动光谱比较 它的瞬时光中间体将有助于阐明结构 这种蛋白质在运作过程中发生的变化。《博大》 时间分辨FTIR差示光谱的灵敏度应允许 检测发色团中的瞬时结构变化，以及 多肽骨架的局部构象改变； 可电离氨基酸侧链的质子化或去质子化；以及 阳离子残基与其非共价相互作用的变化 氯离子或其他阴离子。这些测量的时间分辨率将 在1-10微秒的范围内。同位素标记和突变型HR的光谱 还将获得蛋白质，以便为个体分配角色 氨基酸在各种阴离子结合和释放步骤中。结果是 应允许主动开发详细的机械模型 这一特殊体系的氯离子输运，并推导出 适用于哺乳动物阴离子转运蛋白的一般原则。

The physiological importance of chloride transport across cell membranes has been underscored recently by the demonstration that genetic defects in a single transmembrane protein involved in chloride ion transport cause cystic fibrosis. However, despite such advances in genetic information the detailed mechanisms of chloride transport by membrane proteins are understood only poorly, in large part because of a paucity of basic information about the dynamics of interactions between anions and their binding sites on transport proteins. We will apply time-resolved Fourier transform infrared (TR-FTIR) spectroscopy to the study of the active chloride transport by biological membranes. The model system to be studied is halorhodopsin (hR), a light-driven anion transport protein found in the membranes of several species of archaebacteria. Comparisons of TR-FTIR vibrational spectra from hR and its transient photointermediates will help to elucidate structural changes occurring during the functioning of this protein. The broad sensitivity of time-resolved FTIR difference spectroscopy should allow detection of transient structural changes in the chromophore, as well as localized conformational alterations of the peptide backbone; protonations or deprotonations of ionizable amino acid side chains; and alterations in non-covalent interactions of cationic residues with chloride or other anions. The time resolution of these measurements will be in the range of 1-10 mus. Spectra of isotope-labeled and mutant hR proteins will also be obtained, in order to assign roles for individual amino acids in various anion binding and release steps. The results should allow the development of a detailed mechanistic model for active chloride transport for this particular system, and the derivation of general principles applicable to mammalian anion transport proteins.