Structural Analysis of Protein-Membrane Interaction

项目摘要/摘要 膜的形状和曲率的控制对许多重要的细胞功能是必不可少的，包括细胞 分裂和细胞运动，以及囊泡的萌发和融合。最近的研究表明，这些过程 是由能够感觉、稳定或诱导膜弯曲的蛋白质调节的。不足为奇的是， 膜曲率控制中的异常与许多疾病有关，包括精神疾病 发育迟缓、癌症和肌肉营养不良。然而，潜在的分子机制并不充分。 由于几乎不存在与生物相关的膜结合形式的结构信息，因此人们对 曲率感应或诱导蛋白质。这项提案的中心目标是提供这样详细的结构 和机械信息。 目的1研究膜联蛋白感知膜曲的分子机制。这 非凡的过程导致膜联蛋白由内向外的主要重折叠，并带来埋藏的疏水残基 溶液结构与膜的酰基链直接接触。使用以下组合 连续波和脉冲EPR谱，拟议的工作将定义三维结构 这种膜结合形式，并检验N-末端磷酸化调节曲率的假设- 体内依赖的膜相互作用。此外，还将测试，曲率的结构是否- 依赖的膜结合形式与界面的pH依赖的膜结合形式有关。 目的2研究曲率诱导N-bar蛋白内亲和膜结合形式。 两栖类，而AIM 3则涉及F-bar蛋白FCHo2的膜结合形式的结构。 在这两个目标中提出的详细的结构研究旨在测试BAR域的假设 蛋白质通过三种不同机制的组合诱导细胞膜弯曲：(1)作为 形成特定膜曲率的支架；(2)通过将两亲性螺旋“楔入”膜中； 以及(3)通过形成特定排列的寡聚体结构。特别是，将测试N-bar和 F-bar蛋白通过上述机制在不同程度上诱导弯曲。所有结构 从EPR分析中获得的数据将使用最近开发的PRONOX算法进行进一步改进 它将用于生成三维结构模型，其中包含蛋白质以及 脂膜。这些模型将提供对BAR的分子机制的直接洞察 含有结构域的蛋白质会引起膜弯曲。将进一步使用结构分析来测试 两栖动物蛋白-2突变体(K35N和D151N)降低曲率诱导特性的机制 以及家族性中央核肌病的功能障碍。

PROJECT SUMMARY/ABSTRACT The control of membrane shape and curvature is essential for many vital cellular functions, including cell division and cell motility, as well as vesicle budding and fusion. Recent work has shown that these processes are regulated by proteins that can sense, stabilize or induce membrane curvature. Not surprisingly, aberrations in the control of membrane curvature have been linked to a number of diseases, including mental retardation, cancer and muscular dystrophy. The underlying molecular mechanisms, however, are poorly understood since little structural information exists for the biologically relevant membrane-bound forms of the curvature-sensing or inducing proteins. The central goal of this proposal is to provide such detailed structural and mechanistic information. Aim 1 investigates the molecular mechanisms by which annexins can sense membrane curvature. This remarkable process causes a major inside-out refolding of annexins and brings buried hydrophobic residues of the solution structure into direct contact with the acyl chains of the membranes. Using a combination of continuous wave and pulsed EPR spectroscopy, the proposed work will define the three-dimensional structure of this membrane-bound form and test the hypothesis that N-terminal phosphorylation modulates curvature- dependent membrane interaction in vivo. Furthermore, it will be tested, whether the structure of the curvature- dependent membrane bound forms is related to that of the interfacial, pH-dependent membrane-bound form. Aim 2 investigates the membrane-bound forms of the curvature-inducing N-BAR proteins endophilin and amphiphysin, while aim 3 addresses the structure of the membrane-bound form of the F-BAR protein FCHo2. The detailed structural studies proposed in both aims are designed to test the hypothesis that BAR domain proteins induce membrane curvature using a combination of three different mechanisms: (1) by acting as scaffolds that mold a specific membrane curvature; (2) by "wedging" amphipathic helices into the membrane; and (3) by forming specifically aligned oligomeric structures. In particular it will be tested whether N-BAR and F-BAR proteins induce curvature by using the aforementioned mechanisms to different extents. All structural data obtained from the EPR analysis will be refined further using the recently developed PRONOX algorithm which will be employed to generate three-dimensional structural models that contain the proteins as well as the lipid membranes. These models will provide direct insight into the molecular mechanisms by which BAR domain-containing proteins induce membrane curvature. Structural analysis will further be used to test the mechanism by which amphiphysin-2 mutants (K35N and D151N) have reduced curvature-inducing properties and misfunction in familial forms of centronuclear myopathy.