Signaling Biophysics of Protein-GTPase Interactions

描述（由申请人提供）：轴突引导的抑制或缺乏影响神经元细胞运动性和位置维持，导致神经元再生失败！受伤后的联系有缺陷的指导也与从老年痴呆症到癌症等疾病有关。我们研究的长期目标是确定涉及轴突导向信号受体和小GTP酶的信号转导机制的生物物理基础。丛蛋白-B1跨膜蛋白是第一个直接与小GT3，Rac 1相互作用的受体。该项目的主要目标是提供一个详细的结构和热力学特性的基础上的丛蛋白Rac 1相互作用，并了解其在丛蛋白介导的信号转导的作用。 我们的假设是，填充不同构象状态的倾向已经是Rac 1结合结构域的一个特征，并且如果不是在其与GTP酶的相互作用或响应于其他扰动时，这种调节转换行为在该丛蛋白结构域本身中将是明显的。具体目标：1.确定丛蛋白B1的Rac 1结合结构域的结构，并表征其二聚化界面和推定的Rac 1结合开关。核磁共振（NMR）光谱将用于结构测定的单体形式的丛蛋白，并将被用来映射野生型蛋白质的丛蛋白二聚化界面。2.也在非结合突变体中的丛蛋白Rac 1结合结构域-GTdR界面的表征，以及蛋白丛蛋白-Rac 1复合物的结构的测定。3.将定量由丛蛋白引起的热力学扰动：GTdR络合，并确定所涉及结构的稳定性和结合亲和力之间的关系。全局热力学扰动之后，将荧光和圆二色谱，具体网站的变化酰胺氢交换与NMR光谱。重要性：这些研究将确定涉及小GTP酶-蛋白质相互作用的信号传导机制的生物物理基础，并将为涉及小GTP酶在信号传导事件中的其他相互作用提供更深入的见解。

DESCRIPTION (provided by applicant): Inhibition or lack of axon guidance affects neuronal cell motility and positional maintenance, resulting in the failure to regenerate neurona! connections after injury. Defective guidance is also associated with diseases ranging from Alzheimers to cancer. The long term objective of our research is to determine the biophysical basis of signal transduction mechanisms involving axon guidance cue receptors and small GTPases. The Plexin-B1 transmembrane protein is the first example of a receptor that interacts directly with small GTPase, Rac1. The major goal of the proposed project is to provide a detailed structural and thermodynamic characterization for the basis of the plexin-Rac1 interaction and to understand its role in plexin mediated signal transduction. Our hypothesis is that the propensity to populate different conformational states is already a characteristic of the Rac1 Binding Domain, and that such regulatory switching behavior will be apparent in this plexin domain by itself, if not upon its interaction with the GTPase or in response to other perturbations. Specific aims: 1. Determination of the structure of the Rac1 binding domain of plexin-B1 and characterization of its dimerization interface and of the putative Rac1 binding switch. Nuclear Magnetic Resonance (NMR) spectroscopy will be used for structure determination of a monomeric form of plexin, and will be utilized to map the plexin dimerization interface of the wild type protein. 2. Characterization of the plexin Rac1 binding domain - GTPase interface, also in a non-binding mutant, and determination of the structure of the protein plexin-Rac1 complex. 3. Thermodynamic perturbations caused by plexin:GTPase complexation will be quantified and the relationship between stability and binding affinity of the structures involved will be determined. Global thermodynamic perturbations will be followed by fluorescence and circular dichroism spectroscopy, site specific changes by amide hydrogen exchange in conjunction with NMR spectroscopy. Significance: These studies will determine the biophysical basis for a signaling mechanism involving a small GTPase - protein interaction and will provide deeper insights into other interactions that involve small GTPases in signaling events.