Metalloenzyme Mechanisms

描述(申请人提供)：我们的主要研究目标是阐明金属蛋白体系中的分子识别机制，包括蛋白质与其他蛋白质、小分子底物/抑制剂以及建立和控制超分子膜复合体组装的脂膜成分的识别，这些超分子膜复合体影响识别和催化的各个方面。努力集中在人肝细胞色素P450上，它是药物代谢的核心角色。由于它们在人类健康和疾病中的重要作用，这些酶系统已经占据了研究人员几十年的时间。尽管进行了大量的研究工作，但对P450催化的生物物理和生化机制的完整分子理解仍然是重要的目标。一个障碍是人类细胞色素P450是完整的膜蛋白。虽然通过研究它们在天然膜制剂中的功能或通过洗涤剂的增溶作用已经了解了很多，但关于膜在促进功能方面的作用和底物识别的基础的关键知识还很缺乏。在一种新的方法中，我们通过利用纳米盘系统(在本赠款的支持下开发)克服了这一挑战，该系统将膜蛋白靶标自组装成可溶于水的盘状纳米级脂质双层。我们使用各种生物物理方法来定义蛋白质自组装成这些健壮的双层结构的机制，并揭示P450及其氧化还原伙伴细胞色素P450还原酶(CPR)的拓扑结构。为了了解脂质如何与蛋白质和多蛋白质复合体相互作用的细节，我们定义了人肝脏P450和CPR之间的复合体的形成机制，并探索了P450寡聚在药物代谢中的潜在作用。人类P450解毒酶需要一个高度可塑性的活性部位，以便在功能上适应广泛的底物结构。结合和代谢P450活性部位的多种底物的能力是药物-药物相互作用的根源，在许多情况下，这是在疗效和毒性之间的治疗窗口中产生有害副作用和限制的原因。因此，了解分子识别的这一方面的精确机制和协同底物结合的大小也是我们未来工作的重要目标。这些旨在揭示金属蛋白机制中分子识别原理的努力是通过应用广泛的生物物理、生化和结构技术来揭示自组装的分子细节、膜环境的作用以及细胞色素P450催化的蛋白质构象决定因素。

DESCRIPTION (provided by applicant): Our major research goal is elucidating the mechanisms of molecular recognition in metalloprotein systems, including the recognition of proteins with other proteins, with small molecule substrates/inhibitors and with the lipid membrane components that establish and control the assembly of supra-molecular membrane complexes that affect all aspects of recognition and catalysis. Efforts focus on the human hepatic cytochromes P450 that are central players in drug metabolism. Due to their important role in human health and disease, these enzyme systems have occupied investigators for many decades. Despite an intense research effort, a complete molecular understanding of the biophysical and biochemical mechanisms of P450 catalysis remain important goals. One obstacle has been that the human cytochrome P450s are integral membrane proteins. While much has been learned by investigating their function in native membrane preparations or by detergent solubilization, critical knowledge on the role of the membrane in facilitating function and the basis for substrate recognition is lacking. In a novel approach, we surmount this challenge by making make use of the Nanodisc system (developed under support from this grant) that self-assembles the membrane protein target into a discoidal nanometer size lipid bilayer that is soluble in aqueous solution. We use a variety of biophysical approaches in order to define the mechanism of protein self-assembly into these robust bilayer structures and to reveal the topology of P450 and its redox partner, cytochrome P450 reductase (CPR). In seeking to understand the details of how lipids interact with proteins and multi-protein complexes, we define the mechanisms of complex formation between human hepatic P450 and CPR as well as explore the potential role of P450 oligomerization in drug metabolism. The human P450 detoxifying enzymes require a highly plastic active site in order to functionally accommodate a wide spectrum of substrate structures. The ability to bind and metabolize multiple substrates in the P450 active site is the origin of "drug-drug" interactions, which in many cases, is responsible for deleterious side effects and limitations in the therapeutic window between efficacy and toxicity. Hence, understanding this aspect of molecular recognition in the precise mechanisms and magnitude of cooperative substrate binding is also an important goal of our future work. These efforts to reveal the principles of molecular recognition in metalloprotein mechanisms are linked through application of a breadth of biophysical, biochemical and structural techniques to reveal the molecular details of self-assembly, the role of the membrane environment and protein conformational determinants of cytochrome P450 catalysis.