REGULATION OF G PROTEINS BY MASTOPARAN

Mastoparan 对 G 蛋白的调节

• 批准号: 3298451
• 负责人: TSUTOMU HIGASHIJIMA
• 金额: $ 17.58万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298451
• 关键词: G protein; Hymenoptera; affinity chromatography; affinity labeling; biological signal transduction; chemical binding; circular dichroism; computer assisted sequence analysis; conformation; crosslink; fluorescence spectrometry; laboratory rat; nuclear magnetic resonance spectroscopy; peptide analog; peptide chemical synthesis; protein purification; protein structure function; receptor binding; receptor coupling

Mastoparan, a peptide toxin from wasp venom, causes the activation of GTP- binding regulatory proteins (G proteins) by promoting GTP binding in a manner strikingly similar to that of cell surface receptors. mastoparan (MP), a cationic, amphiphilic helix, is also structurally similar to putative G protein-binding domains on these receptors. I propose to study the mechanism and structural basis of the G protein activation by MP and related amphiphilic compounds as a model for receptor-G protein coupling and to develop G protein-specific regulatory peptides as probes of cellular regulation. (1) We will develop MP analogs that are more potent and more selective G protein activators and inhibitors. Structure-activity analysis will use kinetic assays with purified G proteins and recombinant alpha subunits, physical studies of MP-G protein complexes, and computer assisted modeling. (2) Affinity cross-linking between MP and G proteins will be performed to determine the MP-binding site, presumably the receptor binding-site as well. We will evaluate competition between receptor and MP for binding to G proteins to evaluate the identity of their binding sites. (3) Circular dichroism, 19F-NMR, and fluorescence spectroscopy will be used to study the structural basis of MP-G protein binding. Two-dimensional transferred NOE of 1H-NMR (500 MHz) will be used to determine the conformation of MP when bound to G proteins. The conformational basis of MP binding to G proteins will be compared with its binding to calmodulin, which binds many amphiphilic peptides. (4) We will study the mechanism by which MP and related compounds activate G proteins or block activation using kinetic and ligand binding assays. We will clarify the roles of the G protein betagamma subunits and Mg2+ on MP action. We will extend these studies to small GTP-binding proteins, particularly p21ras, which are not known to be regulated by receptors. (5) MP analogs will be used to study G protein-mediated signaling in cells. Affinity crosslinking of MP targets in cells, measurements of MP uptake and its mechanism and the use of MP as an affinity chromatographic ligand will be included in these experiments.

Mastoparan 是一种来自黄蜂毒液的肽毒素，可激活 GTP- 通过促进 GTP 结合来结合调节蛋白（G 蛋白） 方式与细胞表面受体惊人相似。 马斯托帕兰 (MP)，一种阳离子两亲性螺旋，结构上也类似于 这些受体上假定的 G 蛋白结合域。 我建议学习 MP和G蛋白激活G蛋白的机制和结构基础 相关的两亲性化合物作为受体-G蛋白偶联的模型 并开发G蛋白特异性调节肽作为细胞探针 规定。 (1) 我们将开发更有效、更具选择性的 MP 类似物 G 蛋白质激活剂和抑制剂。 结构-活性分析将使用 使用纯化的 G 蛋白和重组 α 亚基进行动力学测定， MP-G 蛋白复合物的物理研究和计算机辅助建模。 (2) MP和G蛋白之间进行亲和交联以 确定 MP 结合位点，大概是受体结合位点 出色地。 我们将评估受体和 MP 之间的竞争结合 G 蛋白来评估其结合位点的身份。 (3)将使用圆二色性、19F-NMR、荧光光谱 研究 MP-​​G 蛋白结合的结构基础。 二维 1H-NMR (500 MHz) 的转移 NOE 将用于确定 MP 与 G 蛋白结合时的构象。 的构象基础 MP 与 G 蛋白的结合将与其与钙调蛋白的结合进行比较， 它结合许多两亲性肽。 (4) 研究MP及相关化合物的激活机制 使用动力学和配体结合测定法检测 G 蛋白或阻断激活。 我们 将阐明 G 蛋白 betaamma 亚基和 Mg2+ 对 MP 的作用 行动。 我们将把这些研究扩展到小的 GTP 结合蛋白， 特别是 p21ras，目前尚不清楚其是否受受体调节。 (5) MP类似物将用于研究细胞中G蛋白介导的信号传导。 细胞内 MP 目标的亲和交联、MP 摄取和测量的测量 其机制以及 MP 作为亲和色谱配体的用途将 包括在这些实验中。