SITE DIRECTED ISOTOPE LABELING OF MEMBRANE PROTEINS

SIR提供L-[4- 13] C天冬氨酸; 2g L-[1，5_13C 2]谷氨酸; 0.5g FTIR差谱可 用于探测单个氨基酸水平的构象变化 膜蛋白中的酸残基。 然而，一个关键问题限制了 进步是缺乏一个通用的方法， 同位素标记在蛋白质的特定位置。 直到最近 这是不可能的，除非通过化学合成， 通常限于50个残基的多肽。 我们最近 他提出了一种新的方法来避免这种限制。 称为 定点同位素标记（SDIL），它是基于使用 抑制性tRNA和体外合成。 的情况下 细菌视紫红质，光驱动的质子泵， 方法来标记几个酪氨酸和肽羰基位置。 结合FrIR差分光谱，这使我们能够 在Tyr残基185中的精确结构活性，其可用作 用于蛋白质构象变化铰链。 我们现在的目标是 扩展这些方法，以便它们可以用于常规标记任何 细菌视紫红质中的残基;可用于其他蛋白质， 可以与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，新方法将 被开发用于酶促和化学氨基酰化， 抑制性tRNA以优化体外表达、分离和 重折叠和SDIL蛋白的光谱表征。 一个 将产生大量SDIL细菌视紫红质类似物 包括位于Asp、Glu、Pro、Thr、Ser和Trp位置的标记。 这些研究将直接导致一个详细的图片， 蛋白质功能 类似的研究将在感官上进行。 视紫红质I，盐杆菌中的趋光受体。 盐疗室。

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.