EFFECTS OF PREPROTEIN & NUCLEOTIDE BINDING ON SOLUTION STRUCTURE OF SECA ATPASE

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。 赛卡ATPase是原核生物中一种重要的蛋白质转运系统--前蛋白转位酶的引擎。SecA与蛋白质传导孔SecYEG相互作用，并利用ATP的能量将未折叠的前蛋白转移到细胞膜上。为了推动前蛋白通过SecYEG，SecA经历了重复的前蛋白结合和释放周期，这与ATP结合和水解有关。SAXS实验的目标是研究前蛋白和/或核苷酸结合时在SecA中发生的构象变化。 除了是易位引擎外，SecA还是一种伴侣，因为它只与未折叠的前蛋白相互作用，这使得对SecA-前蛋白相互作用的详细生物物理研究变得不可能，因为前蛋白在实验过程中要么会折叠，要么会聚集。为了克服这一技术障碍，我们使用固定化多肽文库来寻找与SecA高亲和力结合的短肽序列。利用这项技术，我们已经鉴定了一些高度可溶的15个残基多肽，并以核苷酸依赖的方式与SecA结合。我们认为，当SecA与这些多肽结合时，它会发生显著的构象变化。 SECA是一种多结构域的模块化蛋白(图1在所附的SecA-SAXS.pdf中)。根据各种结构(图2)和生化数据(未显示)，我们认为前蛋白结合在核苷酸结合域和前蛋白结合域之间，需要在SecA(图3)中的构象变化；图4提供了SAXS中伴随的变化的一个例子。利用SecA的模块化性质，我们有4个不同的结构可用于SAXS实验：前蛋白和核苷酸结合对每个结构的结构的影响将使我们能够绘制发生的域间运动，并显示ATP结合和水解是如何耦合到前蛋白结合的。

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 SecA ATPase is the engine of preprotein translocase, an essential protein translocation system in prokaryotes. SecA interacts with the protein-conducting pore, SecYEG, and uses the energy of ATP to translocate unfolded preproteins across the cytoplasmic membrane. To push the preprotein through SecYEG, SecA undergoes repeated cycles of preprotein binding and release, which are linked to ATP binding and hydrolysis. The goal of the SAXS experiments is to investigate the conformational changes that take place in SecA when preprotein and/or nucleotides are bound. In addition to being an engine for translocation, SecA is a chaperone in the sense that it interacts only with unfolded preprotein, and this has made detailed biophysical studies of the SecA-preprotein interaction impossible because the preprotein will either refold or aggregate during the experiment. To overcome this technical hurdle, we have used immobilized peptide libraries to find short peptide sequences that bind to SecA with high affinity. Using this technology, we have identified a number of 15 residue peptides that are highly soluble and bind to SecA in a nucleotide-dependent manner. We believe that there are significant conformational changes in SecA when it binds these peptides. SecA is a multi-domain, modular protein (Fig. 1 in attached SecA-SAXS.pdf). Based on a variety of structural (Fig. 2) and biochemical data (not shown), we believe the preprotein is bound between the nucleotide binding domains and the preprotein binding domain, requiring conformational changes in SecA (Fig. 3); an example of the attendant changes in SAXS is provided in Fig. 4. Taking advantage of the modular nature of SecA, we have 4 different constructs to use for SAXS experiments: the effect of preprotein and nucleotide binding on the structure of each construct will allow us to map the inter-domain movements that take place and show how ATP binding and hydrolysis are coupled to preprotein binding.