Toxic Metal Complexation By de Novo Designed Peptides

描述(由申请人提供)：尽管重金属毒性和与金属稳态失调相关的神经退行性疾病对人类健康造成了广泛的危害，但对重金属蛋白相互作用的详细分子理解尚未达到。有关有毒金属性质的基本化学问题仍然存在，因为这些体系往往过于复杂、瞬时或不可溶，无法以产生有用信息的方式进行探索。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或死亡的过程。这项应用的总体目标是使用一种创新的方法-从头蛋白质设计，来评估有毒金属与蛋白质结合的热力学和动力学，并提供新的光谱关联，以显著增强有毒金属-蛋白质相互作用的表征。我们的中心假设是，我们定义良好的从头设计的金属蛋白(α-螺旋3链螺旋线圈和3-螺旋束)将提供对有毒金属化学的详细洞察，可用于了解更复杂的系统。我们的基本前提是，从头开始的蛋白质设计提供了简单、高度可控的支架，非常适合提取有毒金属化学的基本信息，并通过系统地研究水环境中的不同配位来获得对分子功能的关键见解。这项拟议的研究的基本原理是，它将提供有关蛋白质与有毒金属相互作用的新信息，而这些信息是其他方法未能仔细审查的。在强大的初步数据的指导下，我们的假设将通过三个具体目标进行验证：1)在设计的蛋白质中制备不对称的金属结合部位；2)使用设计的蛋白质来开发金属-蛋白质相互作用的光谱表征；以及3)使用新的蛋白质设计来表征蛋白质中的有毒金属动力学和热力学。目的1应用三种方法(铅辅助组装、多肽共价键和α螺旋束中固有的不对称性)来获得不对称的金属结合位点。这将使我们能够在AIMS 2和AIMS 3中研究异构性有毒金属位置。AIM 2进一步开发了光谱方法(~(113)Cd核磁共振、~(111)mCd PAC、~(207)铅核磁共振、~(204)m~(PAC)PAC)，然后应用我们的相关性在更复杂的自然体系中确定金属位置。目的3通过分析铅(II)、砷(III)和镉(II)如何被插入到我们设计的多肽中，并比较它们与Fe(III)、Cu(I/II)和Zn(II)的结合常数，来表征有毒金属的热力学和动力学。我们的研究将提供重要的结构表征、结合常数和动力学 在开发探测蛋白质结合的有毒金属方法的同时进行研究。我们的研究意义重大，因为澄清热力学和动力学金属识别过程将对蛋白质如何靶向和创新产生预测能力，因为我们使用了一种非传统的方法，从头开始蛋白质设计，以回答通过对天然生化系统的直接研究或通过合成小分子模型络合物无法完全解决的问题。

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.