Redox-Active and Luminescent Probes for Heme Enzymes

描述(由申请人提供)：Gray和Goodin实验室的综合努力将集中在一系列设计的敏化连接底物(SLS)上，这些底物将用作酶功能的探针、抑制剂的发现以及具有生物医学意义的重要金属蛋白的显像剂。驱动这些研究的两个主要假设：1)当与封闭的设计-反馈循环相结合时，合理地设计蛋白质的结构元件是可行的，以及2)关于重要酶的功能的问题可以使用这样的模型来解决。格雷实验室率先使用SLS探针，允许电子转移(ET)直接进入深埋在地下的蛋白质活性部位。古丁实验室的结果表明，可以通过迭代设计来操纵蛋白质，为小分子创造结合位点。在这项提案中，将使用一种组合的分子设计方法，其中蛋白质和SLS都被设计为自我互补。建议的细胞色素c过氧化物酶中的电子转移途径将被切除，并被氧化还原活性的SLS探针取代，以解决关于特定ET途径的化学性质和背景的问题。SLS探针将与细胞色素C氧化酶的血红素A3/幼体中心的基于蛋白质的模型耦合，提供新的方法来测试关于电子供体如何参与O2还原的新想法。将开发新的SLS探针，用于鉴定酶抑制剂和成像特定的生物靶标。其中一类将被设计成在与蛋白质靶标结合时从发光状态切换到黑暗状态，并在竞争抑制物置换时恢复发光。长期目标是开发异构体特异性探针，用于筛选重要酶的抑制剂，如细胞色素P450和一氧化氮合酶(NOS)。第二类探针将被设计成在与目标结合时从黑暗状态切换到发光状态。这些探针将被设计用来检测一氧化氮合酶的异构体；他们的研究将是我们朝着开发新的工具进行体内蛋白质成像和空间定位的长期目标迈出的关键的第一步。总体而言，这一提议将有助于将结构元素设计成蛋白质框架，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.