STRUCTURAL CHARACTERIZATION OF NON-BIOLOGICAL ATP BINDING PROTEINS

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。 我们的实验室已经进化出一系列非生物的ATP结合蛋白，这些蛋白质是从纯随机序列库中进化而来的。最近对其中一个家族的代表的晶体结构进行了解析，发现它与ATP/ADP和二价锌离子结合在一起。为了选择更强的配体结合和结构稳定性，额外的几轮定向进化产生了一个共同的序列，它与其祖先只有2个氨基酸的差异，显示出与ATP/ADP结合，对底物具有更高的亲和力，并且在GuHCl存在下更耐变性。在不同含量的ATP存在下进行的共结晶研究揭示了各种新的配体结合模式。 我们预计这两个氨基酸替换的贡献将是谨慎的，但它们对新的结构和生化观察的贡献是显著的。为了验证这一假设，我们想要在锌吸收边缘(峰、远端和拐点)进行MAD实验，以获得每个结构及其单个点突变的无偏见和独立的相，以确定这两个突变在我们新的非生物ATP结合蛋白中的结构和功能含义。 这些研究提供了有用的信息，说明自然界是如何选择具有特定配体结合能力的蛋白质，但也进化出了在该配体上催化特定酶反应的能力。此外，这些研究应该使我们能够对所需的必要参数(即蛋白质结构、配体结合和催化支架等)收集重要的见解。用于设计新的、可能更好的配基结合剂和酶。

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. Our laboratory has evolved a family of non-biological ATP binding proteins that have been evolved from a pool of purely random sequences. The crystal structure of a representative of one of these families was recently solved and shown to bind ATP/ADP, and a divalent Zn2+ ion. Additional rounds of directed evolution, designed to select for stronger ligand binding and structural stability, yielded a consensus sequence which differed from its progenitor by only 2 amino acids, was shown to display both ATP/ADP binding, possessed much higher affinity for substrate, and was shown to be much more resistant to denaturation in the presence of GuHCl. Co-crystallization studies carried out in the presence of varying amounts of ATP have revealed a variety of new ligand binding modes. We expect the contribution of the 2 amino acid substitutions to be discreet, yet significant in their contribution to the new structural and biochemical observations. To test this hypothesis, we would like to carry out MAD experiments at the zinc absorption edge (peak, remote, and inflection) to acquire unbiased and independent phases for each construct and their individual point mutants to determine the structural and functional implications these 2 mutations have in our novel, non-biological ATP binding protein. These studies promise useful information into how nature has selected for proteins that have developed the ability for specialized ligand binding, but have also evolved the capability of catalyzing specific enzymatic reactions on that ligand. In addition, these studies should allow us to glean important insight into the necessary parameters required (i.e. protein architecture, ligand binding and catalytic scaffolds, etc.) for design of new, and potentially better, ligand binders, and enzymes.