Toxic Metal Complexation By de Novo Designed Peptides

描述（由申请人提供）：尽管由于重金属毒性和与功能失调的金属稳态相关的神经退行性疾病而对人类健康产生广泛的有害影响，但尚未获得对重金属蛋白相互作用的详细分子理解。关于有毒金属性质的基本化学问题仍然存在，因为这些系统通常太复杂，瞬变或不溶性，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或死亡的过程。本申请的总体目标是使用一种创新的方法，从头蛋白质设计，以评估热力学和动力学的有毒金属结合蛋白质，并提供新的光谱相关性，以提高有毒金属蛋白质相互作用的表征显着。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-链卷曲螺旋和3-螺旋束）将提供详细的了解有毒金属化学，可应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简单的，高度可控的支架非常适合提取有毒金属化学的基本信息，并通过系统地检查水溶性肽环境中的不同配位位点来深入了解分子功能。这项研究的基本原理是，它将提供关于蛋白质-有毒金属相互作用的新信息，这些信息逃避了其他方法的审查。在强有力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测试：1）在设计的蛋白质中制备不对称金属结合位点; 2）使用设计的蛋白质开发金属-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的有毒金属动力学和热力学。目的1采用三种方法（铅辅助组装，共价键的肽，和内在的不对称性内的α-螺旋束），以获得不对称的金属结合位点。这将允许研究目标2和3中的混配型有毒金属位点。目的2进一步发展光谱方法（113 Cd NMR，111 mCd PAC，207 Pb NMR，204 mPb PAC）与我们定义良好的金属网站，然后应用我们的相关性，自信地分配金属网站在更复杂的自然系统。目的3通过分析Pb（II），As（III）和Cd（II）如何插入到我们设计的肽中，并将其结合常数与Fe（III），Cu（I/II）和Zn（II）的结合常数进行比较，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 同时开发探测蛋白质结合的有毒金属的方法。我们的研究意义重大，因为热力学和动力学金属识别过程的澄清将产生对蛋白质如何靶向和创新的预测能力，因为我们正在使用非传统的方法，从头蛋白质设计，来回答无法通过直接研究天然生化系统或合成小分子模型复合物来完全解决的问题。

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.