Structural Analysis of Protein-Membrane Interaction

项目总结/摘要 膜的形状和曲率的控制对于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，以及囊泡出芽和融合。最近的研究表明，这些过程 由能够感知、稳定或诱导膜弯曲的蛋白质调节。毫不奇怪， 控制膜曲率的异常与许多疾病有关，包括精神疾病。 发育迟缓、癌症和肌肉萎缩症。然而，潜在的分子机制却很差， 因为几乎没有结构信息存在的生物学相关的膜结合形式的 曲率感应或诱导蛋白。该提案的核心目标是提供这种详细的结构 机械信息。 目的1研究膜联蛋白感知膜曲率的分子机制。这 一个显著的过程导致膜联蛋白的一个主要的由内而外的重折叠，并将膜联蛋白的疏水残基 溶液结构与膜的酰基链直接接触。结合使用 连续波和脉冲EPR光谱，拟议的工作将定义的三维结构 并检验N-末端磷酸化调节曲率的假设- 依赖于体内的膜相互作用。此外，它将被测试，是否结构的曲率- 依赖性膜结合形式与界面pH依赖性膜结合形式相关。 目的2研究诱导弯曲的N-BAR蛋白的膜结合形式， amphiphysin，而aim 3解决了F-BAR蛋白FCHo 2的膜结合形式的结构。 这两个目标中提出的详细结构研究旨在验证BAR结构域 蛋白质使用三种不同机制的组合诱导膜弯曲：（1）通过充当 塑造特定膜曲率的支架;（2）通过将两亲性螺旋“楔入”膜中; 和（3）通过形成特定排列的低聚结构。特别是，将测试N-BAR和 F-BAR蛋白通过使用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算法进行进一步的改进 其将用于产生包含蛋白质以及蛋白质的三维结构模型。 脂膜这些模型将提供直接洞察的分子机制， 含有结构域的蛋白质诱导膜弯曲。结构分析将进一步用于测试 两性物理蛋白-2突变体（K35 N和D151 N）具有降低的弯曲诱导特性的机制 以及家族性中枢性肌病的功能障碍。

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.