ANNEXIN CRYSTAL STRUCTURE AND PHOSPHOLIPID INTERACTION

膜联蛋白晶体结构和磷脂相互作用

• 批准号: 3468143
• 负责人: BARBARA A SEATON
• 金额: $ 10.31万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468143
• 关键词: Raman spectrometry; X ray crystallography; calcium binding protein; chemical binding; computer simulation; crystallization; electron density; heavy metals; interferometry; isomorphous substitution; ligands; liposomes; membrane reconstitution /synthesis; molecular dynamics; molecular shape; phospholipase inhibitor; phospholipids; physical model; protein structure; solutions

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.

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， 该模型可能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，例如无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。