GROEL VARIANT

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Rye 实验室的总体研究目标是了解 GroEL 如何与其伙伴蛋白 GroES 结合，利用 ATP 水解的能量来促进完全依赖 GroEL 的底物蛋白（所谓的“严格”底物）的蛋白质折叠。 GroEL 和 GroES 都是七重环状低聚物。 GroEL 在其一个开环上捕获折叠中间体，然后结合 ATP 和辅助伴侣蛋白 GroES，从而将底物蛋白封装在封闭的 GroEL-GroES 复合物中。 GroES 是如何完成底物封装的，以及 GroEL 对驱动有效折叠的底物蛋白的作用仍然知之甚少。 Rye 通过结合定点诱变、化学修饰和荧光光谱来解决这些问题（Lin 和 Rye，2004 年；Lin 等人，2008 年）。 先前的研究无法捕获和研究 GroEL-GroES 复合物的预触发状态并不奇怪，因为野生型 GroEL 仅短暂地填充这种变构状态（大约一百毫秒），对于准备操作而言时间太短。 相比之下，我们修改后的 GroEL 变体似乎以相当慢的速度完成这种转变。 我们建议，利用 NCMI 在通过冷冻电镜检查 GroEL 等分子方面取得的技术进步，对这种新的 GroEL 变体进行高分辨率观察，可能会提供我们目前知之甚少的 GroEL-GroES 机器的关键变构状态的前所未有的快照。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall research goal in Rye's lab is to understand how GroEL, in conjunction with its partner protein GroES, utilizes the energy of ATP hydrolysis to promote protein folding of fully GroEL-dependent substrate proteins (so-called "stringent" substrates). Both GroEL and GroES are seven-fold, ring shaped oligomers. GroEL captures a folding intermediate on one of its open rings, then binds ATP and the co-chaperonin GroES, resulting in the encapsulation of the substrate protein within an enclosed GroEL-GroES complex. How sub-strate encapsulation by GroES is accomplished and precisely what GroEL does to a substrate protein that drives productive folding remain poorly understood. Rye has approached these problems using a combination of site-directed mutagenesis, chemical modification and fluorescence spectroscopy (Lin and Rye, 2004; Lin et al., 2008). The inability of previous studies to trap and study a pre-triggered state of the GroEL-GroES complex is not surprising, given that wild-type GroEL populates this allosteric state only transiently (for a hundred millisec or so), much too short a time for ready manipulation. By contrast, our modified GroEL variant appears to move through this transition at a considerably slower rate. We propose that a high-resolution look at this new GroEL variant, exploiting the technical advances the NCMI has made in examining molecules like GroEL by cryo-EM, is likely to provide an unprecedented snap-shot of a critical allosteric state of the GroEL-GroES machine about which we currently know very little.