INSERTION/FOLDING OF POLYTOPIC INTEGRAL MEMBRANE PROTEIN

描述：本项目的总体目标是调查 决定蛋白质形成的脂质-蛋白质-蛋白质相互作用 脂质双层结构。 了解影响 膜蛋白的三维结构很重要，因为它是 仍然难以获得膜蛋白的高分辨率结构， X射线或电子晶体学或固态核磁共振。 一个替代 一种可能性是从它们的氨基预测多面体膜结构 酸序列，但预测方法关键取决于参数 描述了细胞膜上发生的相互作用。 此外，本发明还提供了一种方法， 理解膜蛋白折叠具有实际重要性，因为 它将指导研究人员努力重折叠重组蛋白质， 进行结构和其他研究。 外膜蛋白A（Omp A） 大肠选择大肠杆菌作为这些研究的模型蛋白。 这种蛋白质 被认为在膜中形成8股反平行β桶， 作为膜蛋白折叠的范例， β-结构（例如，孔蛋白或乙酰胆碱受体）。 远程 提出的研究目标是制定折叠的一般规则 和膜蛋白的插入。 本研究的主要目的有六个：（1）研究蛋白质的复性动力学; 将脲变性的OmpA转化为具有各种脂质的脂质模型膜， （2）确定反应速率常数和反应机理 从吸附到天然插入状态的OmpA相互转化， 温度依赖性动力学实验，使用野生型和特异性 设计突变体OmpA;（3）进一步表征OmpA的结构 低温下液相脂质中的中间体折叠 荧光猝灭、近紫外CD和偏振ATR-FTIR光谱;（4） 化学合成并荧光标记所有八种跨膜 β链和四个反平行β环，以确定其 双层中的单个结构以及 它们从缓冲液插入到双层中;（5）共重构两个或更多个分子， 更多的吸附和/或插入形式的这些肽，以研究它们的 不同温度下双层膜的自组装动力学及其结构 温度;（6）制定膜蛋白折叠的一般规则 以及根据目标1至5的结果进行插入。

DESCRIPTION: The overall goal of this project is to investigate lipid-protein-protein interactions that determine the formation of protein structure in lipid bilayers. Understanding the factors that govern the three-dimensional structure of membrane proteins is important because it is still difficult to obtain high resolution structures of membrane proteins by X-ray or electron crystallography or solid-state NMR. An alternate possibility is to predict polytopic membrane structures from their amino acid sequences, but predictive methods depend critically on the parameters describing the interactions that occur in the membrane. In addition, under-standing membrane protein folding is of practical importance because it will guide researchers in their efforts to refold recombinant proteins for structural and other studies. Protein A (Omp A) from the outer membrane of E. coli is chosen as a model protein for these studies. This protein is thought to form an 8-stranded antiparallel beta-barrel in membranes and will serve as a paradigm for the folding of membrane proteins that contain beta-structure (e.g., porins or acetylcholine receptors). The long-range goal of the proposed research is to formulate general rules for the folding and insertion of membrane proteins. There are six specific aims: (1) to study the kinetics of refolding of urea-denatured OmpA into lipid model membranes with various lipids and at various temperatures; (2) to determine the rate constants and the mechanism of OmpA interconversion from the adsorbed to the native inserted state by temperature-dependent kinetic experiments using wild-type and specifically designed mutant OmpAs; (3) to further characterize the structure of OmpA folding intermediates in fluid phase lipid at low temperature by fluorescence quenching, near UV CD, and polarized ATR-FTIR spectroscopy; (4) to chemically synthesize and fluorescently label all eight membrane-spanning beta-strands and the four antiparallel beta-loops to determine their individual structures in bilayers and the thermodynamics and kinetics of their insertion from buffer into bilayers; (5) to co-reconstitute two or more of these peptides in the adsorbed and/or inserted forms to study their kinetics of self-assembly and their structures in bilayers at different temperatures; and (6) to formulate general rules of membrane protein folding and insertion based on the results of aims 1 through 5.