Redox-Active and Luminescent Probes for Heme Enzymes

描述（由申请人提供）：格雷和古丁实验室的综合努力将集中于一系列设计的敏化剂连接底物（SLS），这些底物将用作酶功能的探针、抑制剂的发现以及生物医学上重要的金属蛋白的成像剂。两个主要假设推动了这些研究：1）当与封闭的设计反馈循环相结合时，将结构元件合理设计成蛋白质是可行的；2）可以使用此类模型来解决有关重要酶功能的问题。格雷实验室率先使用 SLS 探针，将电子转移 (ET) 引导至深埋的蛋白质活性位点。古丁实验室的结果表明，可以通过迭代设计来操纵蛋白质，以创建小分子的结合位点。在该提案中，将使用组合分子设计方法，其中蛋白质和 SLS 被设计为自我互补。细胞色素 c 过氧化物酶中拟议的电子转移途径将被切除并替换为氧化还原活性 SLS 探针，以解决有关特定 ET 途径的化学性质和背景的问题。 SLS 探针将与细胞色素 c 氧化酶血红素 a3/CUB 中心的基于蛋白质的模型耦合，为测试有关电子供体如何参与 O2 还原的新兴想法提供新方法。将开发新型 SLS 探针，用于识别酶抑制剂和对特定生物靶标进行成像。一类将被设计为在与蛋白质靶标结合后从发光状态切换到黑暗状态，并在被竞争性抑制剂取代后恢复发光。长期目标是开发异构体特异性探针，用于筛选重要酶（如细胞色素 P450 和一氧化氮合酶 (NOS)）的抑制剂。第二类探针将被设计为在与目标结合后从暗状态切换到发光状态。这些探针将被设计用于检测 NOS 同工型；他们的研究将代表我们朝着开发体内蛋白质成像和空间定位新工具的长期目标迈出关键的第一步。总体而言，该提案将为将结构元件设计到蛋白质框架中提供一种全新的方法，以检测、控制和理解金属蛋白质的行为。

DESCRIPTION (provided by applicant): Integrated efforts of the Gray and Goodin laboratories will focus on a series of designed sensitizer-linked substrates (SLS) to be used as probes of enzyme function, for the discovery of inhibitors, and as imaging agents for biomedically important metalloproteins. Two primary hypotheses drive these studies: 1) rational design of structural elements into a protein is feasible when coupled to a closed design-feedback cycle, and 2) questions about the function of important enzymes may be addressed using such models. The Gray laboratory has pioneered the use of SLS probes, allowing electron transfer (ET) to be directed into deeply buried protein active sites. Results in the Goodin laboratory have shown that proteins may be manipulated by iterative design to create binding sites for small molecules. In this proposal, a combined molecular design approach will be used in which both the protein and the SLS are designed to be self complementary. The proposed electron transfer pathway in cytochrome c peroxidase will be excised and replaced with redox active SLS probes to address questions about the chemical nature and context of specific ET pathways. SLS probes will be coupled to a protein-based model for the heme a3/CUB center of cytochrome c oxidase, providing new ways to test emerging ideas about how electron donors participate in O2 reduction. Novel SLS probes will be developed for identification of enzyme inhibitors and for imaging specific biological targets. One class will be designed to switch from a luminescent to a dark state upon binding to the protein target, and to recover luminescence upon displacement by a competitive inhibitor. The long-term goal is to develop isoform specific probes for screening inhibitors of important enzymes such as cytochrome P450 and nitric oxide synthase (NOS). A second class of probes will be designed to switch from a dark to a luminescent state upon binding to a target. These probes will be designed to detect NOS isoforms; their study will represent a critical first step toward our long-term goal of developing new tools for imaging and spatial localization of proteins in vivo. Overall, this proposal will contribute a completely novel approach to the design of structural elements into protein frameworks for the purpose of detecting, controlling and understanding the behavior of metalloproteins.