A COMBINATORIAL APPROACH FOR THE INVESTIGATION AND DEVELOPMENT OF NEW CALMODULIN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 一系列的生化和结构研究表明，一种独特的结构元件，中央连接子，在赋予钙调蛋白(CaM)低结合特异性方面发挥了重要作用。中央连接区作为分子铰链，使CaM在整个蛋白质中呈现出较大的构象变化，从而呈现出一个由疏水和酸性残基组成的结构动态表面，以识别、结合并最终将钙信号传递到其众多的靶蛋白。为了破译是什么相互作用导致了CaM的低结合特异性，最近的研究重点是试图提高CaM与单个靶分子的结合亲和力。 与这些先前的研究试图通过先验地重新设计特定的结合相互作用来提高结合特异性不同，另一种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双模式方法来创建高质量、多产的CaM中央连接子组合文库，以检验CaM结合亲和力和特异性是该独特区域提供的氨基酸接触和结构可塑性的组合的结果的假设。所选蛋白质的生化特性不仅有助于了解天然CaM的功能，还将验证这种从头开始的蛋白质设计方法在新的蛋白质支架上的应用。此外，这些文库将作为使用高通量筛选和选择获得配体特异性改变的蛋白质的起点。

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.