Structural Analysis of Protein-Membrane Interaction

描述（由申请人提供）：膜形状和曲率的控制对于许多重要的细胞功能至关重要，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，这些过程是由蛋白质，可以感觉，稳定或诱导膜曲率。毫不奇怪，控制膜曲率的异常与许多疾病有关，包括精神发育迟滞、癌症和肌肉萎缩症。然而，基本的分子机制知之甚少，因为几乎没有结构信息存在的曲率传感或诱导蛋白的生物相关的膜结合形式。本提案的中心目标是提供这种详细的结构和机械信息。目的1研究膜联蛋白感知膜曲率的分子机制。这一非凡的过程导致膜联蛋白的主要内翻式重折叠，并使溶液结构中隐藏的疏水残基与膜的酰基链直接接触。使用连续波和脉冲EPR光谱的组合，所提出的工作将定义这种膜结合形式的三维结构，并测试N-末端磷酸化调节体内曲率依赖性膜相互作用的假设。此外，将测试曲率依赖性膜结合形式的结构是否与界面pH依赖性膜结合形式的结构相关。目的2研究了弯曲诱导N-BAR蛋白质endophilin和amphiphysin的膜结合形式，而目的3解决了F-BAR蛋白质FCHo 2的膜结合形式的结构。两个目标中提出的详细结构研究旨在检验BAR结构域蛋白使用三种不同机制的组合诱导膜曲率的假设：（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）通过形成特异性对齐的寡聚体结构。特别地，将测试N-BAR和F-BAR蛋白是否通过使用上述机制在不同程度上诱导弯曲。从EPR分析获得的所有结构数据将使用最近开发的PRONOX算法进一步细化，该算法将用于生成包含蛋白质和脂质膜的三维结构模型。这些模型将提供直接洞察的分子机制，其中BAR结构域的蛋白质诱导膜曲率。结构分析将被进一步用于测试的机制，通过该机制，两栖physin-2突变体（K35 N和D151 N）已减少弯曲诱导性能和功能障碍的家族性形式的中枢性肌病。公共卫生相关性：所有细胞都需要能够控制其膜的形状和曲率。当这个过程不能正常工作时，它会导致癌症，肌肉萎缩症和智力迟钝等疾病。目前的建议旨在建立机制，参与这一过程的蛋白质可以感知和诱导细胞膜的适当曲率。这些结果将对我们理解和治疗上述疾病产生影响。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: All cells need to be able to control the shapes and curvatures of their membranes. When this process is not working properly, it can lead to diseases such as cancer, muscular dystrophy and mental retardation. The present proposal seeks to establish the mechanism by which proteins involved in this process can sense and induce the appropriate curvature of cellular membranes. The results will have implications for our understanding and treatment of the aforementioned diseases.