ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS

膜联蛋白晶体结构和膜相互作用

• 批准号: 2734659
• 负责人: BARBARA A SEATON
• 金额: $ 24.04万
• 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 1999-12-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734659
• 关键词: X ray crystallography; amphiphilicity; annexins; calcium; calcium channel; chemical binding; conformation; crosslink; crystallization; fluorescence spectrometry; hydropathy; lectin; liposomes; membrane activity; membrane proteins; membrane reconstitution /synthesis; nuclear magnetic resonance spectroscopy; phospholipids; phosphorylation; physical model; protein purification; protein structure function; site directed mutagenesis; solutions; structural biology

Proteins that are active at cell membrane-cytosol interfaces operate in a unique, two-phase environment, and their molecular mechanisms represent a current frontier of cell biology. Annexins are a family of interfacial calcium-dependent membrane-binding proteins that are widely distributed in eukaryotes. Found in very large amounts (1-2% total cell protein) in many tissues, annexins exist stably in water-soluble and membrane-bound forms. For structural biologists, annexins present an unusual opportunity to study a myriad of protein-mediated membrane processes. Annexin V, a putative effector of calcium movement across membranes, has provided an excellent structural prototype for study of the annexin family and other interfacial proteins. The long-term goal of this research program is to develop an integrated picture of annexin behavior at the membrane. The primary goal of the proposed research is to develop a molecular model of membrane-bound annexin V, detail its interactions with membrane components, and characterize its effects on membrane parameters. X-ray crystallography and solution methods will be used as complementary approaches to study both water-soluble and membrane-bound forms of annexin V. The specific aims of this proposal are to; 1) Crystallographically study annexin-ligand interactions; 1 a) Refine solved crystal structures with calcium, lipid, and phospholipid analogues bound; 1b) determine crystal structures of annexin V complexes with a series of deacylated or short-chain phosphatidylserine compounds; 1 c) solve structures of non-phospholipid compounds bound to an intermolecular hydrophobic site in annexin V crystals; 1d) determine structures of carbohydrate-annexin V complexes. 2) Investigate annexin V properties by mutagenesis, using crystallography and solution assays (protein-vesicle binding, chemical cross-linking, NMR spectroscopy, fura-2-fluorimetry): 2a) A K yields E mutation in a non- functional calcium-binding loop to see if calcium-binding can be restored; 2b) an E yields V mutation causing loss of calcium binding in an otherwise functional loop, to see the resulting effect on phospholipid head-group binding at this site; 2c) substitution of putative determinants to see whether the lipid binding preference of annexin V can be changes to that of annexin I, to probe the structural basis of phospholipid specificity; 2d) mutations that disrupt trimer formation, to study the relationship between oligomerization and other annexin properties. 3) Crystallize, for further study, two other annexins; 3a) phosphorylated annexin iV, to study the structural consequences of this modification; and 3b) annexin XI, which, unlike annexin V, has an extensive N-terminal domain. Information from these studies will add much to the rather sparse body of knowledge about interfacial proteins, provide a unifying structural basis for annexin action at he membrane, and propose the structural basis for putative annexin V function as it may relate to kidney, lung, liver, and other organ tissues in health and disease.

活跃于细胞膜-细胞质界面的蛋白质以一种独特的方式运作