ANNEXIN CRYSTAL STRUCTURE AND PHOSPHOLIPID INTERACTION

Annexins形成一组同源的胞浆蛋白，结合 磷脂膜以钙依赖的方式。他们一直都是 与多种细胞功能有关，包括分泌、融合和 信号转导。膜联蛋白，其分子量通常为 35 kDa或68 kDa，表现出约70个残基的共同正则序列， 重复了四到八次。这些重复与模式不符 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调节蛋白。 这项研究的长期目标是发展一种分子机制 可能普遍适用于膜联蛋白和其他钙的模型- 依赖的磷脂结合蛋白。中提出的具体目标 这一提议是Annexin V的结构表征，A 具有代表性的膜联蛋白家族成员及其与钙和钙的相互作用 通过X-射线结晶学和拉曼光谱对磷脂配体进行了表征。 晶体结构(S)将提供一个详细的分子模型 蛋白质，而光谱研究将探索 蛋白质-磷脂在溶液中的结合。拉曼光谱将提供 晶体和溶液实验之间的桥梁，样品可以在 任何一种形式。 已经生长出与钙结合的膜联蛋白V的大单晶，这些单晶 适用于X射线晶体结构测定。它们是绕射的 分辨率至少为2.2a，并且属于R3空间群，具有 六方晶胞尺寸a=b=156.8A，c=36.9A。要解决这个问题 相位问题，多重同构替换(MIR)或迭代单 将使用同构替换(ISIR)方法。尝试次数将是 使用。将尝试生长和分析Annexin V晶体 其他配位态，如无钙形式或具有可溶性的 磷脂配体结合。 拉曼光谱提供了有关蛋白质和脂肪的详细信息 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 已经得到了V。这种光谱技术将被用来评估 膜联蛋白-磷脂相互作用对蛋白质结构的影响 以及结合的磷脂，如果能有合适的重组系统 发展起来的。峰值分配将通过使用 含全氚碳氢化合物尾部的磷脂。

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.