STRUCTURAL CHARACTERIZATION OF NON-BIOLOGICAL ATP BINDING PROTEINS

这个子项目是许多研究子项目中的一个 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来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们的实验室已经进化出一个非生物ATP结合蛋白家族，这些蛋白是从一组纯粹随机的序列中进化出来的。 这些家族之一的代表的晶体结构最近被解决，并显示出结合ATP/ADP和二价Zn 2+离子。 设计用于选择更强的配体结合和结构稳定性的另外几轮定向进化产生了与其祖先仅相差2个氨基酸的共有序列，显示出ATP/ADP结合，对底物具有高得多的亲和力，并且显示出在GuHCl存在下对变性具有更高的抗性。 在不同量的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2个氨基酸取代的贡献是谨慎的，但在新的结构和生物化学观察的贡献显着。为了验证这一假设，我们想在锌吸收边缘（峰值、远端和拐点）进行MAD实验，以获得每个构建体及其单个点突变体的无偏和独立相位，以确定这2个突变在我们的新型非生物ATP结合蛋白中的结构和功能意义。 这些研究提供了有用的信息，说明自然界如何选择具有特异性配体结合能力的蛋白质，但也进化了催化该配体上特定酶反应的能力。 此外，这些研究应该使我们能够收集重要的洞察所需的必要参数（即蛋白质结构，配体结合和催化支架等）。用于设计新的、可能更好的配体结合剂和酶。

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.