Toxic Metal Complexation By de Novo Designed Peptides

从头设计的肽与有毒金属络合

基本信息

批准号：
9441799
负责人：
VINCENT L PECORARO
金额：
$ 33.14万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-06-09 至 2020-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9441799
关键词：
Active Sites Address Affinity Alzheimer&apos s Disease Arsenic Binding Binding Proteins Binding Sites Biochemical Biological Process Blood Cadmium Chemicals Chemistry Child Chronic Complex Data Environment Fingers Food Supply Goals Grant Health Health Care Costs Heavy Metals Homeostasis Human Ions Kinetics Lead Ligands Long-Term Effects Malignant Neoplasms Mercury Metal Binding Site Metal exposure Metalloproteins Metals Methods Modeling Molecular Molecular Conformation Morbidity - disease rate Mycobacterium tuberculosis NMR Spectroscopy Nerve Degeneration Parkinson Disease Peptides Plant Roots Poisoning Process Property Protein Engineering Proteins Reaction Research Resolution Site Spectrum Analysis Sulfur System Techniques Testing Thermodynamics acute toxicity aqueous contaminated drinking water design genetic regulatory protein inner city innovation insight lead exposure metal complex metal poisoning metalloregulatory protein molecular recognition mortality preference public health relevance scaffold small molecule societal costs toxic metal

项目摘要

DESCRIPTION (provided by applicant): Despite widespread detrimental human health effects due to heavy metal toxicity and neurodegenerative morbidity associated with dysfunctional metal homeostasis, a detailed molecular understanding of heavy metal protein interactions has yet to be attained. Fundamental chemical questions regarding toxic metal properties remain because these systems are often too complex, transient, or insoluble to be probed in a way that yields useful information. Our long-term goal is to resolve the processes by which toxic metals interact with proteins leading to morbidity or mortality. The overall objective of this applicationis to use an innovative approach, de novo protein design, to assess thermodynamics and kinetics for toxic metals binding to proteins and provide new spectroscopic correlations to enhance characterization of toxic metal-protein interactions significantly. Our central hypothesis is that our well-defined de novo designed metalloproteins (alpha-helical 3-stranded coiled coils and 3-helix bundles) will provide detailed insight into toxic metal chemistry that can be applied to understand more complex systems. Our basic premise is that de novo protein design provides simple, highly-controllable scaffolds well-suited to extract fundamental information on toxic metal chemistry and yield key insight into molecular function by systematically examining different coordination sites in aqueous peptidic environments. The rationale of the proposed research is that it will provide new information on protein-toxic metal interactions that have eluded the scrutiny of other approaches. Guided by strong preliminary data, our hypothesis will be tested through three Specific Aims: 1) Prepare asymmetric metal binding sites in designed proteins; 2) Use designed proteins to develop spectroscopic characterization of metal-protein interactions; and 3) Use new protein designs to characterize toxic metal dynamics and thermodynamics in proteins. Aim 1 applies three approaches (Pb-assisted assembly, covalent linkage of peptides, and inherent asymmetry within an alpha-helical bundle) to obtaining asymmetric metal binding sites. This will allow study of heteroleptic toxic metal sites in Aims 2 and 3. Aim 2 further develops spectroscopic methods (113Cd NMR, 111mCd PAC, 207Pb NMR, 204mPb PAC) with our well-defined metal sites, then applies our correlations to confidently assign metal sites in more complex natural systems. Aim 3 characterizes toxic metal thermodynamics and kinetics by analyzing how Pb(II), As(III), and Cd(II) are inserted into our designed peptides and compares their binding constants with those for Fe(III), Cu(I/II), and Zn(II). Our research will provide vital structural characterization, binding constants, and kinetic studies while developing methods for probing protein-bound toxic metals. Our research is significant because clarification of thermodynamic and kinetic metal recognition processes will yield predictive power over how proteins are targeted and innovative because we are using a non-traditional approach, de novo protein design, to answer questions that cannot be fully addressed by direct studies on native biochemical systems or by synthesizing small molecule model complexes.

描述（由申请人提供）：尽管重金属毒性和神经退行性发病率与功能障碍金属稳态相关，但人类健康的影响很大，但对重金属蛋白质相互作用的详细分子理解尚未达到。关于有毒金属特性的基本化学问题仍然存在，因为这些系统通常太复杂，瞬态或不溶性，无法以产生有用信息的方式进行探测。我们的长期目标是解决有毒金属与蛋白质相互作用导致发病率或死亡率的过程。该应用的总体目标是使用创新方法，从头蛋白设计，以评估与蛋白质结合的有毒金属的热力学和动力学，并提供新的光谱相关性，以增强有毒金属蛋白相互作用的表征。我们的中心假设是，我们从头设计明确的金属蛋白（α-螺旋3链盘绕线圈和3螺旋束）将提供对有毒金属化学的详细见解，可用于了解更复杂的系统。我们的基本前提是，从头蛋白质设计提供了非常适合提取有毒金属化学的基本信息，并通过系统地检查水性肽环境中的不同配位地点来提取有关分子功能的基本信息，从而提供了简单的，高度控制的支架。拟议的研究的理由是，它将提供有关蛋白质毒性金属相互作用的新信息，这些信息已经避免了对其他方法的审查。在强大的初步数据的指导下，我们的假设将通过三个特定目的进行检验：1）在设计蛋白质中准备不对称的金属结合位点； 2）使用设计的蛋白质来发展金属 - 蛋白质相互作用的光谱表征； 3）使用新的蛋白质设计来表征蛋白质中有毒金属动力学和热力学。 AIM 1应用三种方法（PB辅助组件，肽的共价连接以及α-螺旋束中的固有不对称性）来获得不对称的金属结合位点。这将允许在AIM 2和3中研究杂色的有毒金属位点。目标2进一步开发了光谱方法（113CD NMR，111MCD PAC，207pb NMR，204MPB PAC）与我们定义良好的金属位点，然后将我们的相关性应用于更复杂的自然系统中。 AIM 3通过分析如何将PB（II）AS（III）和CD（II）插入我们设计的肽中，并将其与Fe（III），Cu（II/II）和Zn（II）进行比较，AIM 3表征了有毒金属热力学和动力学。我们的研究将提供重要的结构表征，结合常数和动力学研究在开发探测蛋白质结合有毒金属的方法的同时。我们的研究之所以重要，是因为澄清热力学和动力学金属识别过程将产生有关蛋白质的靶向和创新方式的预测能力，因为我们使用了非传统方法，即从头蛋白质设计，以回答无法通过对天然生物学系统或合成小型分子模型复合物的直接研究来完全解决的问题。