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

使用 De Novo 蛋白质模型了解二铁羧基的功能调节

基本信息

批准号：
8689205
负责人：
Amanda Reig
金额：
$ 22.18万
依托单位：
URSINUS COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-05 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8689205
关键词：
Active Sites Affect Affinity Amino Acid Sequence Amino Acids Aromatic Amines Binding Biochemical Biological Biomimetics Charge Chemicals Cleaved cell Comparative Study Computer Simulation Development Disease Electronics Electrons Environment Enzymes Family Ferritin Foundations Funding Future Geometry Goals His-His-His-His-His-His Histidine Hydrogen Bonding Hydrogen Peroxide Hydroquinones Individual Investigation Ions Iron Lead Link Metalloproteins Metals Modeling Modification Molecular Molecular Biology Molecular Medicine Mutation N hydroxylation Nature Outcome Oxygen Peptide Sequence Determination Peroxidases Phenols Play Property Proteins Publishing Relative (related person)Role Scaffolding Protein Series Structural Models Structure Structure-Activity Relationship Techniques Therapeutic Agents Variant Work base carboxylate catalyst chemical reaction cofactor design electronic structure experience improved in vivo inositol oxygenase insight member nitric oxide reductase oxidation peroxidation preference protein structure public health relevance research study scaffold success tool undergraduate student

项目摘要

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.

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、特性和几何形状来控制金属蛋白质的化学反应性。我们目前对这些变异如何以及为何导致功能变化的理解相当有限，极大地抑制了分子医学领域的进步。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配置有相当大的相似性，但它们仍能催化多种具有重要生物学意义的化学反应。为了实现这一目标，我们将通过计算设计、实验生产并全面表征一系列小型非天然模型蛋白质，其中铁配位氨基酸的数量、身份和几何形状系统地变化。我们选择的支架是 DFsc，它是从头设计的二铁羧酸蛋白的 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残基的组合进行协调。 DFsc 折叠良好、热力学稳定且具有催化活性。最近，通过添加单个活性位点组氨酸残基和三个支持突变，该蛋白质的化学反应性从苯酚氧化改变为 N-羟基化。在这项先前工作的基础上，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质，以探索红红菊酯中 H2O2 与 O2 的偏好，(2) 创建活性位点中组氨酸/羧酸盐比例增加的新模型蛋白质确定电荷增加的电子和功能后果，以及（3）研究组氨酸/羧酸盐比率对我们的从头蛋白质模型的金属结合偏好的影响。在这些研究结束时，我们将在分子水平上深入了解控制生物氧化的基于结构的因素。我们的研究结果将为未来开发新型和改进的仿生催化剂和基于蛋白质的治疗剂奠定基础。