Using De Novo Protein Models to Understand Functional Tuning in Di-Iron Carboxyla

描述（由申请人提供）：自然界通过改变蛋白质活性位点中配位氨基酸的数量、身份和几何形状来控制金属蛋白的化学反应性。我们目前对这些变异如何以及为什么会导致功能变化的理解相当有限，这严重阻碍了分子医学领域的进展。该项目的总体目标是了解二铁羧酸酶的结构-功能关系，尽管它们的活性位点构型具有相当大的相似性，但它们催化多种生物学上重要的化学反应。为了实现这一目标，我们将计算设计，实验生产，并全面表征一系列小的，非自然的模型蛋白质，其中铁配位氨基酸的数量，身份和几何形状是系统变化的。我们选择的支架是DFsc，是从头设计的二铁羧酸盐蛋白的due ferri家族的成员，其中两个铁原子通过自组装四螺旋束内的组氨酸和羧酸盐残基的组合来协调。DFsc是良好折叠的、热稳定的和催化活性的。最近，这种蛋白质的化学反应性被改变，从苯酚氧化N-羟基化，通过添加一个单一的活性位点组氨酸残基和三个支持突变。在此之前的工作的基础上，我们的目标是（1）创建具有额外的活性位点羧酸残基的新模型蛋白，以探索红赤藓红蛋白中H2 O2与O2的偏好，（2）创建在活性位点具有增加的His/羧酸比率的新模型蛋白，以确定增加的电荷的电子和功能后果，（3）研究组氨酸/羧酸盐比例对我们的从头蛋白质模型的金属结合偏好的影响。在这些研究的结论，我们将获得分子水平的洞察力，以结构为基础的因素，控制生物氧化。我们的研究结果将为未来开发新的和改进的仿生催化剂和基于蛋白质的治疗剂提供基础。

DESCRIPTION (provided by applicant): Nature controls the chemical reactivity of metalloproteins by varying the number, identity, and geometry of the coordinating amino acids in the protein active site. Our current understanding of how and why these variations lead to functional changes is rather limited, significantly inhibiting progress in the field of molecular medicine. The overall objective of this project is to understand the structure-function relationships in di-iron carboxylate enzymes, which catalyze a diversity of biologically important chemical reactions despite considerable similarities in their active site configurations. To accomplish this goal, we will computationally design, experimentally produce, and comprehensively characterize a series of small, unnatural model proteins in which the number, identity, and geometry of the iron- coordinating amino acids are systematically varied. Our scaffold of choice is DFsc, a member of the due ferri family of de novo designed di-iron carboxylate proteins in which two iron atoms are coordinated by a combination of histidine and carboxylate residues within a self-assembling four-helix bundle. DFsc is well-folded, thermodynamically stable, and catalytically active. Recently, the chemical reactivity of this protein was altered from phenol oxidation to N-hydroxylation by the addition of a single active site histidine residue and three supporting mutations. Building on this prior work, we aim to (1) create new model proteins with additional active site carboxylate residues to explore the H2O2 vs. O2 preference in rubrerythrins, (2) create new model proteins with increased His/carboxylate ratios in the active site to determine the electronic and functional consequences of increased charge, and (3) investigate the influence of the His/carboxylate ratio on the metal-binding preferences of our de novo protein models. At the conclusion of these studies, we will have gained molecular-level insight into the structure-based factors that control biological oxidation. Our results will provide a foundation for the future development of new and improved biomimetic catalysts and protein-based therapeutic agents.