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

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 SecA ATP酶是 发动机 前蛋白转位酶是原核生物中一种重要的蛋白质转位系统。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运穿过细胞质膜。到 推 前蛋白通过SecYEG，SecA经历前蛋白结合和释放的重复循环，这与ATP结合和水解有关。SAXS实验的目的是研究前蛋白和/或核苷酸结合时SecA中发生的构象变化。 除了作为一个 发动机 对于易位，SecA是一种伴侣蛋白，因为它只与未折叠的前蛋白相互作用，这使得SecA-前蛋白相互作用的详细生物物理学研究不可能，因为前蛋白在实验过程中会重新折叠或聚集。为了克服这一技术障碍，我们使用固定化肽文库来寻找以高亲和力结合SecA的短肽序列。使用这种技术，我们已经确定了一些15个残基的肽是高度可溶性的，并结合SecA的核苷酸依赖性的方式。我们相信SecA在结合这些肽时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见随附的SecA-SAXS.pdf）。基于各种结构（图2）和生物化学数据（未显示），我们认为前蛋白结合在核苷酸结合结构域和前蛋白结合结构域之间，需要SecA的构象变化（图3）; SAXS中伴随变化的示例见图4。利用SecA的模块化性质，我们有4种不同的构建体用于SAXS实验：前蛋白和核苷酸结合对每个构建体结构的影响将使我们能够绘制发生的结构域间运动，并显示ATP结合和水解如何与前蛋白结合偶联。

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.