REGULATION OF G PROTEINS BY MASTOPARAN

Mastoparan是一种来自黄蜂毒液的肽毒素，它能激活GTP- 结合调节蛋白（G蛋白），通过促进GTP结合， 与细胞表面受体的方式惊人地相似。 mastoparan (MP)，阳离子，两亲性螺旋，在结构上也类似于 这些受体上的假定G蛋白结合域。 我打算学习 MP激活G蛋白的机制和结构基础， 作为受体-G蛋白偶联模型的相关两亲化合物 并开发G蛋白特异性调节肽作为细胞凋亡的探针 调控 (1)我们将开发更有效和更有选择性的MP类似物， 蛋白质激活剂和抑制剂。 结构活性分析将使用 用纯化的G蛋白和重组α亚单位进行动力学测定， MP-G蛋白复合物的物理研究和计算机辅助建模。 (2)将进行MP和G蛋白之间的亲和交联， 确定MP结合位点，可能是受体结合位点， 好. 我们将评估受体和MP之间结合至 G蛋白，以评估其结合位点的身份。 (3)将使用圆二色谱、19 F-NMR和荧光光谱 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， 其结合许多两亲性肽。 (4)我们将研究MP和相关化合物激活 G蛋白或阻断激活使用动力学和配体结合测定。 我们 将阐明G蛋白β γ亚基和Mg ~（2+）对MP的作用 行动上 我们将把这些研究扩展到小的GTP结合蛋白， 特别是p21 ras，其不知道受受体调节。 (5)MP类似物将用于研究细胞中G蛋白介导的信号传导。 细胞中MP靶标的亲和交联，MP摄取的测量和 其机理和MP作为亲和色谱配体的用途将 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.