SITE DIRECTED ISOTOPE LABELING OF MEMBRANE PROTEINS

SIR提供L-[4-13天冬氨酸；2g L-[1，5_13C2]谷氨酸；0.5g FTIR差示光谱分析 用于在单个氨基酸水平上探测构象变化 膜蛋白中的酸残基。然而，一个关键的问题限制了 进步是缺乏纳入的一般方法 蛋白质中特定位置的同位素标记。直到最近 这是不可能的，除非通过化学合成，这是 一般限于50个残基的多肽。我们最近做了 介绍了一种绕过这一限制的新方法。被称为 定点同位素标记(SDIL)，它是基于使用 抑制因子tRNAs和体外合成。在.的情况下 细菌视紫红质，光驱动的质子泵，我们用这个 方法标记多个酪氨酸和多肽的羰基位置。 结合FrIR-Difference光谱，这使得我们能够 精确定位Tyr残基185的结构活性，可能是 这种蛋白质的构象变化是由铰链决定的。我们现在的目标是 扩展这些方法，以便它们可以用来常规地标记任何 细菌视紫红质中的残留物；可以应用于其他蛋白质，并可以 与固态等生物物理技术结合使用 核磁共振、中子衍射和傅里叶变换红外光谱。为此，新的方法将 可用于酶促和化学芳酰化。 抑制因子tRNAs的体外表达、分离和优化 复性和光谱学鉴定SDIL蛋白。一个 将生产广泛的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。 这些研究将直接导致对这一点的详细了解 蛋白质的功能。类似的研究将在感官方面进行。 视紫红质I，嗜盐细菌的趋光性受体。萨利纳姆。

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.