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

描述（由申请人提供）：大自然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导致功能改变的理解相当有限，这极大地抑制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，二铁羧酸酶催化多种重要的生物化学反应，尽管它们的活性位点构型相当相似。为了实现这一目标，我们将通过计算设计、实验生产和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，它是新设计的二羧酸铁蛋白的铁家族的一员，其中两个铁原子通过组氨酸和羧酸残基在自组装的四螺旋束内的组合来协调。DFsc具有良好的折叠性、热力学稳定性和催化活性。最近，通过添加一个活性位点组氨酸残基和三个支持突变，该蛋白的化学反应活性从苯酚氧化改变为n -羟基化。在此基础上，我们的目标是(1)创建具有额外活性位点羧酸残基的新模型蛋白，以探索红红thrin中H2O2与O2的偏好；(2)创建具有活性位点His/羧酸比增加的新模型蛋白，以确定电荷增加的电子和功能后果；(3)研究His/羧酸比对我们的新蛋白质模型的金属结合偏好的影响。在这些研究的结论中，我们将获得分子水平上对控制生物氧化的基于结构的因素的深入了解。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.