Active Site Modifications in De Novo Designed Metalloenzymes

从头设计金属酶的活性位点修饰

• 批准号: 8433582
• 负责人: Virginia May Cangelosi
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8433582
• 关键词: Acetates; Active Sites; Affect; Binding; Binding Proteins; Binding Sites; Bioinorganic Chemistry; Biological Process; Blood; C-terminal; Carbon Dioxide; Carbonic Anhydrase IV; Chemistry; Complex; Core Protein; Cysteine; Diffusion; Disease; Environment; Escherichia coli; Exhibits; Foundations; Goals; Health; Histidine; Human; Hydration status; Hydrogen; Hydrogen Bonding; Hydrolysis; Hydroxide Ion; Hydroxides; Ions; Ligands; Measures; Metal Binding Site; Metalloproteins; Metals; Methods; Modeling; Modification; One-Step dentin bonding system; Peptides; Problem Solving; Process; Protein Binding; Proteins; Reaction; Regulation; Respiration; Role; Site; Spectrum Analysis; Structural Models; Structure; Substrate Interaction; Testing; Threonine; Work; Zinc; analog; base; carbonate dehydratase; design; expectation; improved; insight; metalloenzyme; protein aminoacid sequence; protein folding; protein structure; protein structure function; scaffold; small molecule

DESCRIPTION (provided by applicant): Over one third of all proteins contain metal ions and understanding how these ions provide structural stability, affect folding, and catalyze reactions is critical to understanding metalloprotein structure and function. Many essential biological processes, such as respiration and hydrolytic chemistry, depend on metalloenzymes. A clear understanding of function is essential for solving problems that arise when the proteins misfunction, causing disease. One metalloenzyme, carbonic anhydrase, uses a Zn(II) ion bound by three histidine residues to hydrate CO2, a process that is critical to human health. The goals of this project are 1) to design a simple peptidic construct containing a three histidine metal binding site, 2) to characterize the metal-bound protein, 3) to measure the reactivity of the metalloprotein toward CO2 hydrolysis, and 4) to improve the reactivity by introducing hydrogen binding sites near the active center. To achieve these goals, de novo design principles will be utilized. A single-stranded anti-parallel three-helix protein containing three histidine residues near the C-terminal end will be expressed in E. coli, purified, and bound to Zn(II), Co(II), and Cd(II). Characterization by NMR, EPR, EXAFS, UV/vis, and CD spectroscopies and XRD will give a thorough description of the binding site environment, which is expected to be structurally similar to that of carbonic anhydrase. Using the Zn(II)-bound construct, the activity toward p-nitrophenyl acetate hydrolysis and CO2 hydration will be measured with the expectation of catalytic activity. Finally, hydrogen bonding residues will be incorporated near the active site which should activate the hydroxide ligand on Zn(II) as Thr199 does in carbonic anhydrase. This study will provide a general method for the incorporation of a reactive metal site into a simplified protein construct, result in a carbonic anydrase mimic, and establish the effects of secondary interactions on protein-bound metals. This will further the field of bioinorganic chemistry by moving it one step closer to its ultimate goal of complete control over protein structure and function.

描述（由申请人提供）：超过三分之一的蛋白质含有金属离子，并了解这些离子如何提供结构稳定性，影响折叠和催化反应对于理解金属蛋白结构和功能至关重要。许多基本的生物学过程，例如呼吸和水解化学，都取决于金属酶。对功能的清晰理解对于解决蛋白质功能不全（引起疾病）时出现的问题至关重要。一种金属酶，碳酸酐酶，使用由三个组氨酸残基结合的锌（II）离子来补充二氧化碳，这一过程对人类健康至关重要。该项目的目标是1）设计一个简单的肽构建体，其中包含三个组氨酸金属结合位点，2）表征金属结合的蛋白，3）测量金属蛋白对CO2水解的反应性，而4）通过引入活跃活跃中心附近的氢结合位点来提高反应性。为了实现这些目标，将利用从头设计原则。单链抗平行的三螺旋蛋白包含C末端附近的三个组氨酸残基的三螺旋蛋白将在大肠杆菌中表达，纯化并与Zn（ii），CO（II）和CD（II）结合。 NMR，EPR，EXAFS，UV/VIS和CD光谱和XRD的表征将对结合位点环境进行彻底描述，该环境预计与碳酸酐酶的结构相似。使用Zn（II）结合构建体，将通过催化活性的预期来测量乙酸水解和二氧化碳水解和二氧化碳水解的活性。最后，将在活性位点附近掺入氢键残基，该残基应像Thr199在碳酸酐酶中一样激活锌（II）上的氢氧化物配体。这项研究将提供一种将反应性金属位点掺入简化蛋白质构建体中的一般方法，从而导致碳氧化酶模仿，并确定二级相互作用对蛋白质结合金属的影响。这将通过将其完全控制蛋白质结构和功能的最终目标移动一步，从而进一步进一步进一步。