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

• 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
• 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

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）：自然界通过改变蛋白活性位点配位氨基酸的数量，身份和几何形状来控制金属蛋白的化学反应性。我们目前对这些变化导致功能变化的方式以及为什么的理解相当有限，从而显着抑制了分子​​医学领域的进步。该项目的总体目的是了解Di-Iron羧酸盐酶的结构 - 功能关系，尽管其主动位点构型的相似之处相似，但它们却催化了生物学上重要的化学反应的多样性。为了实现这一目标，我们将在计算设计上，实验生产和全面地表征一系列小型，不自然的模型蛋白，其中铁坐标氨基酸的数量，身份和几何形状是系统地变化的。我们选择的脚手架是DFSC，DFSC是从头设计的Di-Iron羧酸盐蛋白的育狂家族的成员，其中两个铁原子通过组氨酸和羧酸盐残基在一个自组装的四个螺旋束中的组合进行了协调。 DFSC折叠良好，热力学稳定且催化活性。最近，通过添加单个活性位点组氨酸残基和三个支持突变，从苯酚氧化到N-羟基化的化学反应性改变了N-羟基化。 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型号。在这些研究的结论中，我们将获得分子水平的洞察力，以了解控制生物氧化的基于结构的因素。我们的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的基础。