Mechanisms of Visual Signaling

描述（由申请人提供）：本研究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，我们提出了表征恢复蛋白与视紫红质激酶相互作用的实验。这一建议是基于上一个资助期的初步实验，在该实验中，当Ca2+激活的recoverin结合到视紫红质激酶的完整RGS结构域或激酶的n端肽片段时，形成了以前未知的recoverin构象。重要的是，这种新的构象似乎作为一个次要物种（RvCaB）存在于平衡混合物（RvCaA/RvCaB）中，这种混合物是在Ca2+结合恢复蛋白时形成的，即使在没有激酶的情况下。我们的方法将是结合生物物理技术（主要是核磁共振波谱）和体外条件下纯化组分的生化分析来揭示这两种蛋白质相互作用的结构和作用机制的细节。本提案的具体目标有三点：1.具体目标：完成RvCaB的结构表征；2. 通过诱变研究，探讨回收物中哪些氨基酸残基有助于RvCaB的稳定；和3。在原子细节上确定恢复蛋白/视紫红质激酶复合体的结构，最初的重点是激酶的RGS结构域。公共卫生相关性：本研究将在三个方面产生影响：1。了解参与蛋白-蛋白相互作用的信号分子的基本机制；2. 肿瘤相关视网膜病变和细胞增殖异常恢复蛋白表达的研究和3。了解神经元钙传感器蛋白的一般机制。

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.