GROEL VARIANT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Rye实验室的总体研究目标是了解GroEL如何与其合作蛋白GROES一起利用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在它的一个开环上捕获一个折叠中间体，然后与ATP和辅助伴侣Groes结合，导致底物蛋白被包裹在一个封闭的GroEL-Groes复合体中。GroES是如何完成底物封装的，以及GroEL对推动高效折叠的底物蛋白质到底做了什么，仍然知之甚少。黑麦利用定点突变、化学修饰和荧光光谱的组合解决了这些问题(Lin和Rye，2004；Lin等人，2008)。 以前的研究无法捕捉和研究GroEL-Groes复合体的预触发状态并不令人惊讶，因为野生型GroEL只短暂地(100毫秒左右)填充这种变构状态，太短了，无法立即进行操作。相比之下，我们修改后的GroEL变体似乎以相当慢的速度通过了这一过渡。我们建议，利用NCMI在用冷冻EM检查像GroEL这样的分子方面取得的技术进步，以高分辨率观察这个新的GroEL变体，可能会提供一个前所未有的GroEL-Groes机器关键变构状态的快照，目前我们对此知之甚少。

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.