REGULATION OF G PROTEINS BY MASTOPARAN

Mastoparan是一种来自黄蜂毒液的多肽毒素，可激活GTP-1。 通过促进细胞内GTP结合结合调节蛋白(G蛋白) 与细胞表面受体的方式惊人地相似。马斯托瓦兰 (MP)是一种阳离子、两亲性螺旋，结构上也类似于 推测这些受体上的G蛋白结合区。我提议学习 MP和Mp激活G蛋白的机制和结构基础 相关两亲性化合物作为受体-G蛋白偶联的模型 并开发G蛋白特异性调节多肽作为细胞内的探针 监管。 (1)我们将开发更强大、更具选择性的MP类似物 蛋白质激活剂和抑制剂。结构-活性分析将使用 纯化的G蛋白和重组α亚基的动力学分析， MP-G蛋白复合体的物理研究和计算机辅助建模。 (2)MP和G蛋白之间将进行亲和交联，以 确定MP结合部位，推测受体结合部位为 井。我们将评估受体和MP之间的竞争结合 G蛋白，以评估其结合部位的特性。 (3)将使用圆二色谱、~(19)F-核磁共振和荧光光谱 研究MP-G蛋白结合的结构基础。二维图 将使用转移的1H-核磁共振(500 MHz)的NOE来确定 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， 它结合了许多两亲性多肽。 (4)研究MP及其相关化合物的活化机理 G蛋白或阻断激活使用动力学和配基结合分析。我们 将阐明G蛋白β亚基和镁离子在MP中的作用 行动。我们将把这些研究扩展到小的GTP结合蛋白， 尤其是p21ras，它们不受受体的调节。 (5)MP类似物将用于研究G蛋白介导的细胞内信号转导。 MP靶标在细胞中的亲和交联，MP摄取的测量和 其作用机制及MP作为亲和层析配基的应用 被包括在这些实验中。

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.