FUNCTIONAL DESIGN AND REDESIGN OF CA BINDING PROTEINS

提出了一个全面的钙位设计计划，以直接解决该计划项目的主要目标：弥合结构描述和对金属蛋白活性的理解之间的差距。我们研究的广泛和长期目标是开发在生物医学研究和医疗应用中在蛋白质中创建新的功能钙位点的能力。我们项目的一个主要目标是了解EF-手钙结合蛋白(CaBP)对钙结合反应的变化。这些蛋白之所以被选中，是因为它们在几乎所有的钙信号通路中都起着核心作用，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，将使用对EF-Hand CaBP的深入比较分析来提出关于什么结构因素和氨基酸性质控制对钙结合的反应的假说。目标2涉及使用来自目标1的信息来制定关于相互作用的假设，这些相互作用对于稳定不同构象的EF-Hand CaBPs是重要的，然后通过定点突变实验来验证这些假设。这些研究的最终目的是设计、生产和鉴定钙调蛋白，这是一种钙结合蛋白D/9k突变体，其对钙结合的反应已被改变，以模拟同源EF-Hand CABP钙调蛋白的反应。稳定性、钙亲和力和疏水亲和力的测量将用于分析突变体，同时还将使用核磁共振进行结构筛选。功能钙位点设计的最终目标是能够在靶蛋白骨架中插入一个钙位点，但目前还没有这样的方法。在目标3中，我们将实施基于结构的方法，使用程序DEZYMER将金属位点设计为绿色荧光蛋白和光活性黄色蛋白。在开发新型钙生物传感器的背景下，将通过生产和表征结合钙的突变体来测试和改进该方法。我们的结果将有助于整个计划努力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.