INSERTION/FOLDING OF POLYTOPIC INTEGRAL MEMBRANE PROTEIN

描述：这个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 脂双层中的结构。了解制约经济增长的因素 膜蛋白的三维结构很重要，因为它 膜蛋白的高分辨结构仍然很难通过 X射线或电子结晶学或固体核磁共振。替补 根据氨基酸预测多面体膜结构是可能的。 酸序列，但预测方法严重依赖于参数 描述了膜中发生的相互作用。此外, 理解膜蛋白折叠具有重要的实际意义，因为 它将指导研究人员努力重新折叠重组蛋白质 用于结构和其他研究。外膜蛋白A(OMP A) 被选为这些研究的模型蛋白质。这种蛋白质是 认为在膜中形成8链反平行的β-桶，并将 作为包含以下物质的膜蛋白折叠的范例 β-结构(例如，孔蛋白或乙酰胆碱受体)。长距离的 拟议研究的目标是制定折叠的一般规则 以及膜蛋白的插入。 具体目标有六个方面：(1)研究蛋白质的复性动力学 尿素变性的OmpA在不同脂质的脂质模型膜中的应用 不同的温度；(2)测定速率常数和机理 OMPA从吸附状态到天然插入状态的相互转化 利用野生型和特异性的温度相关动力学实验 设计突变体OmpA；(3)进一步鉴定OmpA的结构 低温下液相脂中中间体的折叠 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材料 β链和四个反平行的β环来确定它们的 双层膜中的单层结构及其热力学和动力学 它们从缓冲液插入双分子层；(5)共同重组两个或 更多这些以吸附和/或插入形式存在的多肽用于研究它们的 不同温度下双层膜的自组装动力学及其结构 (6)制定膜蛋白折叠的一般规则 以及基于目标1至5的结果的插入。

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.