A COMBINATORIAL APPROACH FOR THE INVESTIGATION AND DEVELOPMENT OF NEW CALMODULIN

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 一系列的生化和结构研究表明，一个独特的结构元件，中央接头，在赋予钙调素（CaM）低结合特异性中起着重要作用。中央连接区作为一个分子铰链，允许钙调素在整个蛋白质中呈现大的构象变化，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到其众多的靶蛋白。试图破译什么样的相互作用是负责钙调素低结合特异性最近集中在试图提高钙调素对一个单一的目标分子的结合亲和力。 与这些先前的研究相反，这些研究试图通过先验地重新设计特定的结合相互作用来增加结合特异性，不同的方法是通过产生组合蛋白质文库来探索结合亲和力/特异性。拟议的研究将涉及使用二进制模式化的方法对创建高品质的，生产性的组合库的钙调蛋白的中央连接器，以测试的假设，钙调蛋白结合亲和力和特异性是一个组合的结果的氨基酸接触和结构的可塑性提供了这个独特的区域。所选蛋白质的生化表征不仅有助于理解天然钙调素的功能，而且还将验证这种从头蛋白质设计方法对新蛋白质支架的应用。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。

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.