SITE DIRECTED ISOTOPE LABELING OF MEMBRANE PROTEINS

膜蛋白的定点同位素标记

• 批准号: 6309035
• 负责人: KENNETH J ROTHSCHILD
• 金额: $ 2.74万
• 依托单位: UNIVERSITY OF CALIF-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2002-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6309035
• 关键词: bacteria; biological products; biomaterials; biomedical resource; biotechnology; proteins; spectrometry; technology /technique

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.

SIR提供了l- [4-13旋转甲酸； 2G L- [1,5_13C 2]谷氨酸； 0.5G FTIR差异光谱可以是 用于探测单个氨基水平的构象变化 膜蛋白中的酸残基。 但是，关键问题限制 进展是没有一般方法来掺入 同位素标记在蛋白质中的特定位置。 直到最近 除了通过化学合成，这是不可能的 通常仅限于50个残基的多肽。 我们最近有 介绍了一种规避此限制的新方法。 称为 位置定向的同位素标签（SDIL），它基于使用 抑制剂TRNA和体外合成。 如果是 细菌视紫红质，光驱动的质子泵，我们已经使用了 标记几种酪氨酸和肽羰基位置的方法。 结合FRIR差异光谱，这使我们得以 在Tyr残基185中查明结构活动，可以用作 铰链用于该蛋白质的构象变化。 我们的目标是 扩展这些方法，以便它们可以定期标记任何 细菌淡季中的残留物；可以应用于其他蛋白质，可以 与生物物理技术（例如固态）一起使用 NMR，中子衍射和FTIR。 为此，新方法将 开发用于酶促和化学砷化 抑制trNA以优化体外表达，分离和 从光谱上重新塑造和表征SDIL蛋白。 一个 将产生大量的SDIL细菌紫红质类似物 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有关如何的详细描述 蛋白质功能。 对感官的类似研究 Rhodopsin I，盐酸杆菌中的光性受体。 盐业。