OPTICAL STUDIES OF PROTEIN STRUCTURE/FUNCTION

一类肉豆蔻酰化化合物的结构和作用机制 以视网膜恢复蛋白为代表的钙敏感蛋白将是 通过荧光、核磁共振和自旋标签光谱进行了研究。这些 酰化的蛋白质通过结合钙从胞浆移动到膜上。 目的是为了确定钙-肉豆蔻的分子机制。 开关，并确定它们在信号转导过程中的作用。拥有 解决了无钙肉豆蔻酰化产物的三维结构 恢复素和钙结合的非肉豆蔻酰化恢复素，我们现在转向 肉豆蔻酰化钙结合型结构的测定 恢复以揭示开关的精确结构基础。我们的 工作假说是恢复素和脑同系物，如 神经钙素和海马素将钙级联作用于G蛋白 通过与去激活七螺旋的激酶相互作用而级联 感受器。钙离子结合中恢复素的膜接触部位 状态和深度将由自旋标记ESR光谱确定 使用氮氧化物标记的肉豆蔻酸酯和氮氧化物标记的半胱氨酸 通过诱变引入的。结构、光谱和功能 钙离子结合恢复素与视紫红质激酶相互作用的研究 而这一目标的碎片也将被执行。三个班级 将产生并分析恢复素突变体，以进一步 对该传感器的结构和动力学的理解：(1)非极性 肉豆蔻基结合袋中的残基将被极性残基取代。 (2)假定铰链区的甘氨酸将变为丙氨酸。(3) 凹形疏水表面周围的带电残留物，假定 靶结合部位，会带电到带相反电荷的残基。 还将进行神经钙素和海马素的结构研究 这些大脑同源物的靶标将使用酵母进行鉴定 双杂交系统。酵母同源物将被表达和分析以 洞察这一神经细胞钙家族的进化 传感器。

The structure and mechanism of action of a family of myristoylated calcium-sensing proteins, exemplified by retinal recoverin, will be investigated by fluorescence, NMR, and spin-label spectroscopy. These acylated proteins move from the cytosol to a membrane on binding calcium. The aim is to determine the molecular mechanism of calcium-myristoyl switches and define their roles in signal transduction processes. Having solved the three-dimensional structures of calcium-free myristoylated recoverin and calcium-bound unmyristoylated recoverin, we turn now to the determination of the structure of the calcium-bound form of myristoylated recoverin to reveal the precise structural basis of the switch. Our working hypothesis is that recoverin and brain homologs such as neurocalcin and hippocalcin serve to couple calcium cascades to G-protein cascades by interacting with kinases that deactivate seven-helix receptors. The membrane-contact sites of recoverin in the Ca2+-bound state and their depth will be determined by spin-label ESR spectroscopy using nitroxide-labeled myristate and nitroxide-tagged cysteines introduced by mutagenesis. Structural, spectroscopic, and functional studies of the interaction of Ca2+-bound recoverin with rhodopsin kinase and fragments of this target will also be carried out. Three classes of recoverin mutants will be generated and analyzed to further our understanding of the structure and dynamics of this sensor: (1) Nonpolar residues in the myristoyl binding pocket will be replaced by polar ones. (2) Glycines in putative hinge regions will be changed to alanine. (3) Charged residues surrounding a concave hydrophobic surface, the putative target-binding site, will be charged to oppositely-charged residues. Structural studies of neurocalcin and hippocalcin will also be carried out and targets of these brain homologs will be identified using the yeast two-hybrid system. A yeast homolog will be expressed and analyzed to provide insight into the evolution of this family of neuronal calcium sensors.