ANNEXIN CRYSTAL STRUCTURE AND PHOSPHOLIPID INTERACTION

膜联蛋白形成一组同源的胞质蛋白， 磷脂膜以钙依赖的方式。 他们一直 参与多种细胞功能，包括分泌、融合和 信号转导 膜联蛋白，通常具有分子量为 35 kDa或68 kDa，表现出约70个残基的共同典型序列， 重复四到八次。 这些重复不符合 预测钙调蛋白的“E-F手”结构域特征 钙结合蛋白超家族，不结合磷脂。 因此，膜联蛋白代表了一组新的钙介导蛋白。 这项研究的长期目标是开发一种分子机制， 模型，可普遍适用于膜联蛋白和其他钙- 依赖磷脂结合蛋白。 所述的具体目标 这一建议是膜联蛋白V的结构特征， 膜联蛋白家族的代表性成员，及其与钙和 通过X射线晶体学和拉曼光谱学鉴定磷脂配体。 晶体结构将提供晶体的详细分子模型。 蛋白质，而光谱研究将探测的影响， 溶液中的蛋白质-磷脂结合。 拉曼光谱将提供 晶体和溶液实验之间的桥梁，样品可以在 任何形式。 已经生长了钙结合膜联蛋白V的大单晶， 适用于X射线晶体结构测定。 他们会演戏 分辨率至少为2.2A，属于R3空间群， 六方晶胞尺寸a=B= 156.8 A和c= 36.9 A。 解决 相位问题，多同构替换（MIR）或迭代单 将使用同晶替代（ISIR）方法。 尝试将 采用 将尝试生长和分析膜联蛋白V晶体， 其他配体状态，如无Ca 2+形式或具有可溶性 磷脂配体结合。 拉曼光谱提供了蛋白质和脂质的详细信息 构象，并将用于研究配体诱导的构象 膜联蛋白的变化Ⅴ.膜联蛋白的高质量初步拉曼光谱 V已获得。 这种光谱技术将用于评估 膜联蛋白-磷脂相互作用对蛋白质结构的影响， 和结合的磷脂，如果合适的重构系统可以 开发 峰值分配将通过使用 具有完全氘代烃尾的磷脂。

Annexins form a group of homologous cytosolic proteins that bind phospholipid membranes in a calcium-dependent manner. They have been implicated in a variety of cell functions, including secretion, fusion, and signal transduction. Annexins, which typically have molecular weights of 35kDa or 68kDa, exhibit a common canonical sequence of about 70 residues, repeated four or eight times. These repeats do not fit the pattern predicted for "E-F hand" domains characteristic of the calmodulin superfamily of calcium-binding proteins, which do not bind phospholipids. As such, annexins represent a novel group of calcium-mediating proteins. The long-term goal of this research is to develop a molecular mechanistic model that may be generally applicable to annexins and other calcium- dependent phospholipid-binding proteins. the specific aims addressed in this proposal are the structural characterization of annexin V, a representative annexin family member, and its interactions with calcium and phospholipid ligands through X-ray crystallography and Raman spectroscopy. The crystal structure(s) will provide a detailed molecular model of the protein, while the spectroscopic studies will probe the effects of the protein-phospholipid binding in solution. Raman spectroscopy will provide a bridge between crystal and solution experiments, where samples can be in either form. Large single crystals of Ca2+-bound annexin V have been grown that are suitable for X-ray crystallographic structure determination. They diffract to at least 2.2A resolution and belong to the R3 space group, with hexagonal unit cell dimensions of a=b=156.8A and c=36.9A. To solve the phase problem, a multiple isomorphous replacement (MIR) or iterative single isomorphous replacement (ISIR) approach will be used. Attempts will be used. Attempts will be made to grow and analyze annexin V crystals in other liganded states, such as the Ca2+-free form or with a soluble phospholipid ligand bound. Raman spectroscopy provides detailed information on protein and lipid conformation, and will be used to study ligand-induced conformational changes in annexin V. A high-quality preliminary Raman spectrum of annexin V has been obtained. This spectroscopic technique will be used to evaluate the effects of annexin-phospholipid interactions on the protein structure, and the bound phospholipid, if a suitable reconstitution system can be developed. Peak assignments will be facilitated by the use of phospholipids with fully deuterated hydrocarbon tails.