Toxic Metal Complexation By de Novo Designed Peptides

从头设计的肽与有毒金属络合

• 批准号: 8625864
• 负责人: VINCENT L PECORARO
• 金额: $ 29.35万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-09 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8625864
• 关键词: Active Sites; Acute; Address; Affinity; Alzheimer' s Disease; Arsenic; Binding; Binding Sites; Biochemical; Biological Process; Blood; Cadmium; Chemicals; Chemistry; Child; Chronic; Complex; Data; Environment; Fingers; Food Supply; Goals; Grant; Health; Heavy Metals; Homeostasis; Human; Ions; Kinetics; Lead; Ligands; Long-Term Effects; Malignant Neoplasms; Mercury; Metal Binding Site; Metal exposure; Metalloproteins; Metals; Methods; Modeling; Molecular; Morbidity - disease rate; Mycobacterium tuberculosis; NMR Spectroscopy; Nerve Degeneration; Parkinson Disease; Peptides; Plant Roots; Poisoning; Process; Property; Protein Binding; Proteins; Reaction; Research; Resolution; Site; Spectrum Analysis; Sulfur; System; Techniques; Testing; Thermodynamics; Toxic effect; aqueous; cost; design; drinking water; genetic regulatory protein; inner city; innovation; insight; metal poisoning; metalloregulatory protein; molecular recognition; mortality; preference; public health relevance; scaffold; small molecule; toxic metal

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 application is 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 (¿-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 ¿-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.

尽管重金属毒性和神经退行性引起的宽度有害人类健康的影响 与功能失调的金属稳态相关的发病率，对重金属的详细分子理解 蛋白质相互作用尚未附加。有关有毒金属特性的基本化学问题 之所以保持之所 有用的信息。我们的长期目标是解决有毒金属与蛋白质相互作用的过程 导致发病率或死亡率。该应用程序的总体目的是使用创新方法， Novo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 新的光谱相关性可显着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（€螺旋3链盘绕线圈） 和3螺旋捆）将提供有关有毒金属化学反应的详细见解，可以应用于理解 更复杂的系统。我们的基本前提是，从头蛋白设计提供了简单的，高度控制的 脚手架非常适合提取有关有毒金属化学的基本信息，并产生关键的见解 分子功能通过系统地检查彼得腹部水性环境中的不同配位位点。 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 避免了对其他方法的审查。在强大的初步数据的指导下，我们的假设将是 通过三个特定目的进行测试：1）在设计蛋白质中准备不对称的金属结合位点； 2）使用 设计蛋白质以发展金属 - 蛋白质相互作用的光谱表征； 3）使用新 蛋白质设计以表征蛋白质中有毒金属动力学和热力学。 AIM 1应用三个 方法（PB辅助组装，肽的共价连接，并在hellical中继承不对称性 束）获得不对称的金属结合位点。这将允许研究杂色的有毒金属位点 目标2和3。目标2进一步开发光谱方法（113CD NMR，111MCD PAC，207pb NMR，204MPB PAC）使用我们定义明确的金属位点，然后将相关性应用于自信地分配金属位点。 复杂的自然系统。通过分析如何瞄准3个字符的有毒金属热力学和动力学 pb（ii），AS（iii）和Cd（ii）被插入我们设计的肽中，并将其结合常数与 那些用于Fe（III），Cu（I/II）和Zn（ii）。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用于探测蛋白质结合有毒金属的方法时。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 蛋白质的靶向和创新方式的预测能力，因为我们正在使用非传统 方法，从头蛋白质设计，回答直接研究无法完全解决的问题 天然生化系统或通过合成小分子模型复合物。