FUNCTIONAL DESIGN AND REDESIGN OF CA BINDING PROTEINS

提出了一个全面的计划，在钙网站设计直接解决这个程序项目的主要目标：弥合差距之间的结构描述和理解的金属蛋白质的活性。我们研究的广泛，长期目标是开发在蛋白质中创建新的功能性Ca位点的能力，用于生物医学研究和医学应用。我们的项目的一个主要目标是了解EF-手钙结合蛋白（CaBPs）的钙结合反应的变化。选择这些蛋白质是因为它们在几乎所有的Ca信号通路中起核心作用，因此与对健康和疾病（细胞周期，癌症等）的广泛影响有关。在目标1中，深入比较分析EF对健康和疾病（细胞周期，癌症等）的影响范围。在目标1中，深入比较分析EF-手钙结合蛋白将被用来开发什么样的结构因素和氨基酸特性控制响应钙结合的假说。目的2涉及使用的信息，从目的1发展的相互作用是重要的EF-手CaBP的稳定不同的构象的假设，然后通过定点诱变实验来测试这些假设。这些研究的最终目标是设计、生产和表征钙结合蛋白调节蛋白，钙结合蛋白D/9 k突变体，其对钙结合的反应已被改变以模拟同源EF-手CaBP钙调蛋白。稳定性、Ca亲和力和疏水亲和力的测量将用于测定突变体，沿着使用NMR的结构筛选。功能性Ca位点设计的最终目标需要在靶蛋白框架中插入Ca位点的能力，但是这样的方法还不可用。在目标3中，我们将使用程序DEZYMER实现基于结构的方法，将金属位点设计到绿色荧光蛋白和光敏黄色蛋白中。该方法将通过生产和表征结合钙的突变体进行测试和改进，在开发新型钙生物传感器的背景下。我们的研究结果将有助于整个项目的努力，以改善蛋白质能量学和蛋白质设计的耦合，并更好地理解蛋白质中金属位点设计的关键共同原则，包括金属识别，结合和特异性，突变的可加性以及蛋白质环境的长期影响。

A comprehensive program in Ca site design is proposed to directly address the primary objective of this program project: bridging the gap between description of structures and comprehension of activity of metalloproteins. The broad, long-term objective of our research is to develop the ability to create new functional Ca sites in proteins for biomedical research and medical applications. One primary objective of our project is to understand the variation in the responses to Ca binding of EF-hand Ca-binding proteins (CaBPs). These proteins have been selected because they have central roles in nearly all Ca signaling pathways and consequently, are associated with a wide-range of effects on health and disease (cell cycle, cancer, etc.). In Aim 1, in-depth comparative analyses of EF-range of effects on health and disease (cell cycle, cancer, etc.). In Aim 1, in-depth comparative analysis of EF-hand CaBPs will be used to develop hypotheses about what structural factors and amino acid properties control the response to Ca binding. Aim 2 involves using the information from Aim 1 to develop hypotheses about interactions that are important for stabilizing different conformations of EF-hand CaBPs, then testing these hypotheses by site-directed mutagenesis experiments. The ultimate goal of these studies is to design, produce and characterize calbindomodulin, a calbindin D/9k mutant whose response to Ca binding has been altered to mimic that of the homologous EF-hand CaBP calmodulin. Measurements of stability, Ca affinity, and hydrophobic affinity will be used to assay mutants, along with a structural screen using NMR. The ultimate goal of functional Ca site design requires the ability to insert a Ca site in a target protein framework, but such methods are not yet available. In Aim 3, we will implement a structure-based approach using the program DEZYMER to design metal sites into green fluorescent protein and photoactive yellow protein. The method will be tested and refined by producing and characterizing mutants that bind Ca, within the context of developing novel Ca biosensors. Our results will contribute to Program-wide efforts to improve the coupling of protein energetics and protein design, and to better understand key common principles for metal site design in proteins, including metal recognition, binding and specificity, additivity of mutations, and long range effects from the protein environment.