SITE DIRECTED ISOTOPE LABELING OF MEMBRANE PROTEINS

SIR提供了L-[4- 13 Cl天冬氨酸； 2克 L-[1,5_13C 2]谷氨酸； 0.5g FTIR差值光谱可 用于探测单个氨基酸水平的构象变化 膜蛋白中的酸性残基。 然而，一个关键问题限制 进步是缺乏一个通用的方法来纳入 同位素标记在蛋白质的特定位置。 直到最近 除非通过化学合成，否则这是不可能的，即 一般限于50个残基的多肽。 我们最近有 引入了一种新方法来规避此限制。 术语 定点同位素标记（SDIL），它基于使用 抑制 tRNA 和体外合成。 如果是 细菌视紫红质，光驱动质子泵，我们已经使用了它 标记几个酪氨酸和肽羰基位置的方法。 与 FrIR 差光谱相结合，我们能够 精确定位 Tyr 残基 185 的结构活性，该残基可作为 该蛋白质构象变化的铰链。 我们现在的目标是 扩展这些方法，以便它们可以用于常规地标记任何 细菌视紫红质残留；可以应用于其他蛋白质并且可以 与生物物理技术（例如固态技术）结合使用 NMR、中子衍射和 FTIR。 为此，新方法将 被开发用于酶促和化学氨酰化 抑制 tRNA 以优化体外表达、分离和 重折叠并通过光谱表征 SDIL 蛋白。 安 将生产大量 SDIL 细菌视紫红质类似物 包括放置在 Asp、Glu、Pro、Thr、Ser 和 Trp 位置的标签。 这些研究将直接带来关于如何实现这一点的详细描述。 蛋白质功能。 类似的研究将在感官上进行 视紫质 I，盐杆菌中的趋光受体。 盐池。

The SIR provided L-[4- 13 ClAspartic Acid; 2g L-[1,5_13C 2]Glutamic Acid; 0.5g FTIR difference spectroscopy can be used to probe conformational changes at the level of individual amino acid residues in a membrane protein. However, a key problem limiting progress is the absence of a general method for the incorporation of isotope labels at specific positions in a protein. Until recently this was not possible except through chemical synthesis, which is generally limited to polypeptides of 50 residues. We have recently introduced a new method which circumvents this limitation. Termed site-directed isotope labeling (SDIL), it is based on the use of suppressor tRNAs and in vitro synthesis. In the case of bacteriorhodopsin, the light- driven proton pump, we have used this method to label several tyrosine and peptide carbonyl positions. Combined with FrIR-difference spectroscopy, this has allowed us to pinpoint structural activity in Tyr residue 185 which may serve as a hinge for conformational changes in that protein. Our goal is now to extend these methods so that they can be used to label routinely any residue in bacteriorhodopsin; can be applied to other proteins and can be used in conjunction with biophysical techniques such as solid state NMR, neutron diffraction and FTIR. For this purpose new methods will be developed for both enzymatic and chemical arninoacylation of suppressor tRNAs to optimize in vitro expression, isolation and refolding and to characterize SDIL proteins spectroscopically. An extensive set of SDIL bacteriorhodopsin analogs will be produced including labels placed in Asp, Glu, Pro, Thr, Ser and Trp positions. These studies will directly lead to a detailed picture of how this protein functions. Similar research will be conducted on sensory rhodopsin I, the phototactic receptor in Halobacterium. salinarium.