Mechanisms of Visual Signaling

描述(申请人提供)：这项工作的长期目标是阐明脊椎动物光响应信号转导的分子机制。在目前的应用中，我们提出了一些实验来描述恢复素与视紫红质激酶的相互作用。这一建议是基于上一次授权期的初步实验，当时，当钙激活的恢复素与视紫红质激酶的完整RGS结构域或该激酶的N端肽片段结合时，形成了以前未知的恢复素构象。重要的是，这种新的构象似乎以次要物种(RvCaB)的形式存在于平衡混合物(RvCaA/RvCaB)中，该混合物是在没有激酶的情况下与钙离子结合形成的。我们的方法将是结合生物物理技术(主要是核磁共振光谱)和生化分析与体外条件下纯化的成分来揭开这两种蛋白质相互作用的结构和作用机制的细节。这项建议有三个具体的目标：1.完成RvCaB的结构特征；2.通过突变研究，探索RvCaB中哪些氨基酸残基对RvCaB的稳定起作用；3.从原子细节上确定RvCaB/视紫红质激酶复合体的结构，最初的重点是该激酶的RGS结构域。公共卫生相关性：这项研究将在三个方面产生影响：1.了解蛋白质-蛋白质相互作用中涉及的信号分子的基本机制；2.了解癌症相关视网膜病变和细胞增殖中Recovery in的异常表达；3.了解神经元钙感受器蛋白的一般机制。

DESCRIPTION (provided by applicant): The long-term goal of this work is to elucidate molecular mechanisms of vertebrate photoresponse signal transduction. In the current application we propose experiments to characterize the interaction of recoverin with rhodopsin kinase. This proposal is based upon preliminary experiments from the last grant period in which a previously unknown conformation of recoverin was formed when Ca2+-activated recoverin was bound to either the full RGS domain of rhodopsin kinase or an N-terminal peptide fragment of the kinase. Importantly, this new conformation appears to be present as a minor species (RvCaB) in an equilibrium mixture (RvCaA/RvCaB) formed upon the binding of Ca2+ to recoverin even in the absence of the kinase. Our approach will be to use a combination of biophysical techniques (primarily NMR spectroscopy) and biochemical analyses with purified components under in vitro conditions to unravel details of both the structure and mechanism of action for the interaction of these two proteins. There are three Specific Aims in this proposal: 1. To complete structural characterization of RvCaB; 2. To probe through mutagenesis studies which amino acid residues in recovern contribue to stabilization of RvCaB; and 3. To determine in atomic detail the structure of the recoverin/rhodopsin kinase complex with initial focus on the RGS domain of the kinase. PUBLIC HEALTH RELEVANCE: This study will have an impact in three areas: 1. Understanding fundamental mechanisms of signaling molecules involved in protein-protein interactions; 2. Understanding of aberrant recoverin expression for cancer-associated retinopathy and cell proliferation; and 3. Understanding general mechanisms of neuronal calcium sensor proteins.