A COMBINATORIAL APPROACH FOR THE INVESTIGATION AND DEVELOPMENT OF NEW CALMODULIN

研究和开发新钙调蛋白的组合方法

• 批准号: 7720902
• 负责人: Luke H Bradley
• 金额: $ 7.37万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720902
• 关键词: Affinity; Amino Acids; Binding; Biochemical; Calcium Signaling; Calmodulin; Computer Retrieval of Information on Scientific Projects Database; Development; Elements; Funding; Generations; Grant; Hydrophobic Surfaces; Institution; Investigation; Libraries; Ligands; Molecular; Pattern; Play; Proteins; Range; Research; Research Personnel; Resources; Role; Scaffolding Protein; Source; Specificity; Testing; United States National Institutes of Health; combinatorial; design; high throughput screening; improved

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A range of biochemical and structural studies suggest that a unique structural element, the central linker, plays a major role in conferring the calmodulin (CaM) low binding specificity. The central linker region serves as a molecular hinge, allowing CaM to assume large conformational changes throughout the protein, in order to present a structurally dynamic surface of hydrophobic and acidic residues to recognize, bind, and ultimately transmit the calcium signal to its numerous target proteins. Attempts to decipher what interactions are responsible for the CaM low binding specificity have recently focused on trying to improve CaM binding affinity towards a single target molecule. In contrast to these previous studies, which attempted to increase binding specificity by redesigning particular binding interactions a priori, a different approach is to explore binding affinity/specificity through the generation of combinatorial protein libraries. The proposed research would involve using the binary patterning approach towards the creation of high-quality, productive combinatorial libraries of the CaM central linker to test the hypothesis that CaM binding affinity and specificity are a result of a combination of amino acid contacts and structural plasticity provided by this unique region. Biochemical characterization of selected proteins would not only contribute to the understanding of the native CaM's function, but will also validate the application of this de novo protein design approach towards a new protein scaffold. In addition, these libraries would serve as a starting point for obtaining proteins with altered ligand specificities using high throughput screens and selections.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 一系列生化和结构研究表明，独特的结构元件（中央连接体）在赋予钙调蛋白 (CaM) 低结合特异性方面发挥着重要作用。中央连接区充当分子铰链，使 CaM 在整个蛋白质中呈现较大的构象变化，以呈现疏水性和酸性残基的结构动态表面，以识别、结合并最终将钙信号传递至其众多靶蛋白。破译哪些相互作用导致 CaM 低结合特异性的尝试最近集中于尝试提高 CaM 对单个靶分子的结合亲和力。 与这些先前的研究相比，这些研究试图通过预先重新设计特定的结合相互作用来增加结合特异性，不同的方法是通过生成组合蛋白库来探索结合亲和力/特异性。拟议的研究将涉及使用二元模式方法创建高质量、高产的 CaM 中心连接子组合文库，以测试 CaM 结合亲和力和特异性是氨基酸接触和结构可塑性组合的结果的假设由这个独特的地区。所选蛋白质的生化表征不仅有助于理解天然 CaM 的功能，而且还将验证这种从头蛋白质设计方法在新蛋白质支架中的应用。此外，这些文库将作为使用高通量筛选和选择获得具有改变的配体特异性的蛋白质的起点。