Toxic Metal Complexation By de Novo Designed Peptides

尽管重金属毒性和神经退行性疾病对人类健康造成了广泛的有害影响 与金属动态平衡失调相关的发病率，对重金属的详细分子理解 蛋白质的相互作用还有待于实现。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、太短暂或太难溶解，不能以一种能够产生 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。 导致发病或死亡的。此应用程序的总体目标是使用一种创新的方法，即 Novo蛋白质设计，评估有毒金属与蛋白质结合的热力学和动力学，并提供 新的光谱相关性显著增强了有毒金属-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(螺旋三股螺旋卷曲 和3-螺旋束)将提供对有毒金属化学的详细洞察，可用于理解 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简单、高度可控的 适合提取有毒金属化学的基本信息并提供关键洞察力的支架 通过系统研究水环境中不同的配位位置来实现分子功能。 这项拟议研究的基本原理是，它将提供有关蛋白质与有毒金属相互作用的新信息。 避免了其他方法的审查。在强劲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)在设计的蛋白质中制备不对称的金属结合部位；2)使用 设计蛋白质以开发金属-蛋白质相互作用的光谱表征；以及3)使用新的 蛋白质设计用来描述蛋白质中有毒的金属动力学和热力学。目标1适用于三个 方法(铅辅助组装、多肽的共价键和螺旋内固有的不对称性 束)以获得不对称的金属结合部位。这将使研究异感有毒金属部位成为可能 目标2和目标3。目标2进一步发展了波谱方法(~(113)Cd核磁共振、~(111)MCd PAC、~(207)Ph)、~(204)m~( PAC)，然后应用我们的相关性在更多的 复杂的自然系统。目标3通过分析有毒金属的热力学和动力学 将Pb(II)、As(III)和Cd(II)插入到我们设计的多肽中，比较它们的结合常数 铁(III)、铜(I/II)、锌(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，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，因为对热力学和动力学金属识别过程的澄清将产生 对蛋白质如何定位和创新的预测能力，因为我们使用的是非传统的 方法，从头开始蛋白质设计，以回答通过直接研究不能完全解决的问题 天然生化系统或合成小分子模型络合物。

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.