Designing Functional Metalloproteins

金属蛋白质是关键的细胞的主力，催化困难，但必不可少的转变，是必要的 其功能多样，如能量产生、代谢或信号传递。蛋白质结构之间的相互作用 这些酶的催化活性构成了我们工作的基础。蛋白质可能会影响第一个 配位球（配体类型、数量和相对几何形状）以及外部配位环境。 尽管这种化学的不可或缺的性质，一个基本的理解之间的关系， 金属离子催化剂和蛋白质支架仍然难以捉摸。虽然天然金属酶的研究提供了宝贵的 由于这些系统的复杂性，对这些系统的解释往往是不够的， 存在多个金属中心。同样，小分子可以提供反应的洞察力，但往往是有限的 由溶剂或合成的复杂性。出于这个原因，我们利用了一个中间的方法与从头设计 β-螺旋蛋白质易于合成，水溶性，重现天然金属蛋白活性位点， 好吧，并提供类似于本机的折叠，可以简单地操作以限制探测的变量 在我们的研究中。我们的长期目标是以完全的化学分辨率描述金属 与由双螺旋蛋白组成的三股卷曲螺旋（3SCC）相互作用，以获得特定的金属 结构，并提供所需的催化。总体目标是将金属插入到明确的支架中 然后可以改变来测试重要的假设，这些假设将解释这些催化中心如何工作。我们 中心假设是，定义明确的从头金属蛋白可以用来询问金属行为， 以天然蛋白质或小分子难以实现的方式设计蛋白质。我们的基本 前提是从头蛋白质设计提供了简单、高度可控和易于修饰的支架， 非常适合提取金属化学的基本信息。这些结构提供了框架， 检查和比较在水性肽环境中对不同配位位点的系统扰动， 这通常难以用小分子模型，尤其是那些不溶于水的小分子模型来实现。的理由 拟议的研究是，它将提供蛋白质-金属相互作用的新信息， 由于自然系统的复杂性或不溶性，对其他方法的审查。这些研究最终将 形成开发具有不同功能的新催化剂的基础，使用基本上相同的蛋白质配体但不同的金属。 我们现在还可以探讨不对称的第一和第二协调领域环境的影响，并改变 螺旋扭曲对蛋白质的结构和催化行为的影响。我们的假设将通过三个具体目标进行检验： 1)现有3SCC中金属中心的催化活性评价; 2）首次评价 由插入序列不连续性引起的对催化活性的修饰;和3）使用我们的突破 制备重金属模板化的异源三聚体3SCC以产生不对称金属催化剂。这 研究是重要的和创新的，因为它确定了优化生物分子催化的新方法。

Metalloproteins are critical cellular workhorses, catalyzing difficult but essential transformations that are required for functions as diverse as energy generation, metabolism, or signaling. The interplay between protein structure and catalytic activity of these enzymes forms the foundation of our work. The protein may influence both the first coordination sphere (ligand type, number, and relative geometry) as well as the outer coordination environment. Despite the indispensable nature of this chemistry, a fundamental understanding of the relationship between metal ion catalysts and a protein scaffold remains elusive. While native metalloenzyme studies provide valuable insights, interpretations of these systems are often inadequate because of the intricacy of the scaffold or the presence of multiple metal centers. Similarly, small molecules can afford insight on reactions but are often limited by the solvent or synthetic complexity. For this reason, we utilize an intermediate approach with de novo design of -helical proteins which are easily synthesized, water soluble, recapitulate native metalloprotein active sites well, and provide native-like folds that can be manipulated simply in order to limit the variables that are probed during our studies. Our long-term goal is to describe to full chemical resolution the processes by which metals interact with three stranded coiled-coils (3SCC) composed of -helical proteins in order to achieve specific metal structures and afford the desired catalysis. The overall objective is to insert metals into well-defined scaffolds that can then be altered to test important hypotheses that will explain how these catalytic centers work. Our central hypothesis is that well-defined de novo metalloproteins can be used to interrogate metal behavior within designed proteins in ways that would be difficult to achieve with native proteins or small molecules. Our basic premise is that de novo protein design provides simple, highly-controllable, and easily modified scaffolds that are well suited to extract fundamental information on metal chemistry. These structures provide a framework to examine and compare systematic perturbations to different coordination sites in aqueous peptidic environments, which is often difficult to do with small molecule models, especially those insoluble in water. The rationale of the proposed research is that it will provide new information on protein-metal interactions that have eluded the scrutiny of other approaches due to the complexity or insolubility of the natural systems. These studies will eventually form the basis for developing new catalysts with different functions using essentially the same protein ligands but differentmetals. We can also now explore the influence of asymmetric first and second coordination sphere environments and changing the helical twist on the structural and catalytic behavior of proteins. Our hypothesis will be tested through three Specific Aims: 1) Evaluation of catalytic activity for metal centers within existing 3SCC; 2) Assessing for the first time modifications to catalytic activity caused by insertion of sequence discontinuities; and 3) Using our breakthrough preparation of heavy metal templated heterotrimeric 3SCCs to generate asymmetric metal catalysts. This research is significant and innovative as it identifies new approaches to optimize biomolecular catalysis.