Artificial Metalloproteins: Confining Metal Complexes within Protein Hosts. (Orig

这项研究计划的主要目的是确定金属蛋白中活性部位的结构和功能之间的基本关系。这一协作性补充应用建立在关于金属离子的氢键空穴模体(2 RO1 GM050781-21)的现有研究成果的基础上，通过提供一种新的方法，将合成和生物化学与分子生物学相结合，创建控制金属离子周围微环境(次级配位球)的人造金属蛋白。蛋白宿主链霉亲和素和亲和素是合成金属络合物的新结合部位：这些蛋白通常不与金属离子结合，但对生物素具有异常高的亲和力(Ka~1013M-1)。这种特定的结合将被用来将生物素化的合成金属络合物定向到蛋白质中的特定位置。蛋白质宿主将通过非共价相互作用，特别是氢键(H键)来控制金属络合物周围的微环境。这种综合方法的优点是能够独立地调节人造金属辅因子(通过化学方法)和宿主(通过分子生物学方法)的性质，以便容易地提供关于基本结构-功能关系的信息。化学和遗传(化学发生)相结合的方法允许获得一组不同的人造金属蛋白，这些人工金属蛋白可以直接解决活性中心结构如何创建关于金属离子的特定氢键网络的问题。我们的方法允许将两个不同的金属络合物限制在蛋白质内固定(但接近)的位置，因为二级配位球是系统调节的。因此，我们可以探索金属-金属距离和氢键网络的差异如何影响氧气的结合和激活，包括检测通常难以观察到的高能瞬变。长期目标包括发展金属辅助氧化催化的结构和功能关系。 金属蛋白发挥的功能在其他类型的系统中尚未实现，包括人工 金属蛋白。我们的假设是，缺乏对次级协调领域的控制是实现预期功能的主要障碍。来自结构生物学的结果表明，金属蛋白二级配位球内的非共价相互作用在调节功能中起着重要作用。因此，与健康相关的金属蛋白的功能和功能障碍可以在其微环境变化的背景下被理解。即使在生物分子中，非共价相互作用如何能够影响金属介导的过程仍然不清楚。对这些影响的研究需要进行基本的结构、功能和机理研究，在这些研究中，可以单独分析各个组件的影响。我们提出了一种新的化学发生方法，通过这种方法，可以很容易地完成对蛋白质宿主和合成金属络合物结构的特定部位的调节，以便建立与功能的相关性。这些类型的研究将导致对生化过程的基本见解。最终，这项研究将为生物催化剂的性质提供洞察力，并导致新型人工催化剂的出现，这些人工催化剂包含了对金属酶反应特性的精细控制。

The broad purpose of this research program is to determine the fundamental relationships between the structures of active sites in metalloproteins and function. This collaborative supplement application builds on the existing research efforts in Hydrogen Bonding Cavity Motifs about Metal Ions (2 Ro1 GM050781-21) by offering a new approach that combines both synthetic and biological chemistries with molecular biology to create artificial metalloproteins that control the microenvironments (secondary coordination sphere) surrounding metal ions. Protein hosts streptavidin and avidin serve as new binding sites for synthetic metal complexes: these proteins do not normally bind metal ions but have an unusually high affinity for biotin (Ka ~ 1013 M-1). This specific binding will be used to direct biotinylated synthetic metal complexes to specific locations within the proteins. The protein hosts will control the microenvironments around the metal complexes through non-covalent interactions, particularly hydrogen bonds (H-bonds). The strength of this integrated approach is the ability to independently tune the properties of the artificial metal cofactors (through chemical methods) and the hosts (through molecular biology methods) to readily provide information on essential structure-function relationships. The combined chemical and genetic (chemogenetic) approach allows access to a diverse group of artificial metalloproteins that can directly address questions on how active site structures create specific H-bonding networks about metal ions. Our approach allows for the confinements of two distinct metal complexes at fixed (but close) locations within the proteins, as the secondary coordination sphere is systematically modulated. We can thus explore how differences in metal-metal distances and H-bonding networks affect dioxygen binding and activation, including detecting high-energy transients that are often difficult to observe. Long-term goals include developing structure function relationships in metal-assisted oxidative catalysis. Metalloproteins perform functions not yet achieved in other types of systems, including artificial metalloproteins. Our hypothesis is that the lack of control of the secondary coordination sphere is a major obstacle to desired functions. Results from structural biology show that non-covalent interactions within the secondary coordination spheres of metalloproteins are instrumental in regulating function. Therefore the function and dysfunction of health-related metalloproteins can be understood in the context of changes in their microenvironments. It is still unclear, even in biomolecules, how non-covalent interactions are able to influence metal-mediated processes. Investigations into these effects require basic structural, functional, and mechanistic studies in which the effects of individual components can be analyzed separately. We propose a new chemogenetic approach whereby site-specific modulations in structure of the protein host and synthetic metal complex can be readily accomplished, in order to establish correlations with function¿ these types of studies will lead to fundamental insights into biochemical processes. Ultimately, this research will provide insights into the properties of biological catalysts and lead to new classes of artificial catalysts that incorporate the exquisite control of reactivity characteristic of metalloenzymes.