Single-Molecule Study of Dynamics and Mechanisms of Biomacromolecule Interactions

描述（由申请人提供）：为了维持正常代谢，生物体使用复杂的机制来介导细胞内金属离子的安全运输，并调节基因表达以对抗金属诱导的化学应激。然而，所涉及的生物大分子实现其功能的方式在很大程度上是未知的。我们的长期目标是通过利用当前和开发新的单分子荧光方法来了解生物大分子如何共同作用于细胞内金属转运和金属调节，作为了解金属代谢相关疾病原因的先决条件。在这个提议中，我们专注于（1）人类铜伴侣Hah 1如何与铜转运ATP酶威尔逊病蛋白（WDP）相互作用，以实现铜的转运;（2）MerR家族金属调节剂如何与DNA相互作用并改变DNA结构，以实现金属响应性转录调节。我们的具体目标是：1.定义铜运输的Hah 1-WDP相互作用的动力学和机制。在这里，我们将使用纳米囊泡捕获，使单分子FRET研究瞬时蛋白质-蛋白质相互作用。我们的子目标是：1）表征Hah 1和WDP的单金属结合结构域（MBD）如何相互作用以进行铜的运输。2)表征Hah 1和WDP的多结构域构建体如何相互作用以进行铜运输。3)表征WDP内分子内结构域间相互作用的动力学及其与Hah 1铜运输相互作用的耦合。2.定义MerR家族调控因子与DNA相互作用对金属响应转录调控的动力学和机制。我们已经开发了工程化的DNA霍利迪连接（HJ）作为敏感和特异性的单分子报告蛋白质-DNA相互作用。我们的子目标是：1）开发，表征和应用工程化的HJ来报告MerR家族调节因子-DNA相互作用。2)探针MerR家族调节因子施加的DNA解旋用于使用工程化HJ的转录激活。3)使用工程化HJ探测三级调节子-RNA聚合酶-DNA相互作用。这些研究将提供深入了解金属转运蛋白如何合作，以提供金属离子和金属调节剂如何作用于DNA调节转录。该研究中开发的单分子方法将为生物医学研究提供新的实验，并应广泛影响复杂蛋白质-蛋白质和蛋白质-DNA相互作用网络的定量研究。PI符合NIH对新研究者的定义，有资格参与实施缩短新研究者R 01申请的审查周期。公共卫生相关性：这项研究将（1）深入了解细胞内铜转运蛋白的动力学，并为理解铜转运相关疾病的原因提供基础知识，（2）阐明MerR家族调节因子如何控制转录以响应金属离子，并有助于我们理解人类的金属调节。开发的单分子方法将使新的实验能够解决许多生物学问题，并将广泛影响健康相关问题的定量研究，包括金属稳态的生物大分子研究。

DESCRIPTION (provided by applicant): To maintain normal metabolism, organisms use complex machineries to mediate safe trafficking of metal ions inside cells and to regulate expression of genes against metal-induced chemical stress. The ways in which the involved biomacromolecules achieve their functions, however, are largely unknown. Our long-term goal is to understand how biomacromolecules work together for intracellular metal transport and metal regulation by harnessing current and developing new single-molecule fluorescence methods, as a prerequisite for understanding the causes of metal metabolism related diseases. In this proposal, we focus on (1) how human copper chaperone Hah1 interacts with the copper transporting ATPase Wilson disease protein (WDP) for copper trafficking and (2) how MerR-family metalloregulators interact with and change the structure of DNA for metal-responsive transcriptional regulation. Our specific aims are to: 1. Define dynamics and mechanism of Hah1-WDP interactions for copper trafficking. Here we will use nanovesicle trapping to enable single-molecule FRET studies of transient protein-protein interactions. Our subaims are to: 1) Characterize how Hah1 and single metal-binding domains (MBDs) of WDP interact for copper trafficking. 2) Characterize how Hah1 and multi-domain constructs of WDP interact for copper trafficking. 3) Characterize the dynamics of intramolecular interdomain interactions within WDP and their coupling to interactions with Hah1 for copper trafficking. 2. Define the dynamics and mechanism of MerR-family regulator-DNA interactions for metal-responsive transcriptional regulation. We have developed engineered DNA Holliday junctions (HJs) as sensitive and specific single-molecule reporters for protein-DNA interactions. Our subaims here are to: 1) Develop, characterize, and apply engineered HJs to report MerR-family regulator-DNA interactions. 2) Probe MerR-family regulator-imposed DNA unwinding for transcriptional activation using engineered HJs. 3) Probe tertiary regulator-RNA polymerase-DNA interactions using engineered HJs. These studies will provide insight into how metal transporters collaborate to deliver metal ions and how metalloregulators act on DNA to regulate transcription. The single-molecule methods developed in the study will enable new experiments for biomedical research and should impact broadly on quantitative investigations of complex protein-protein and protein-DNA interaction networks. The PI meets the NIH definition of a new investigator and is eligible to participate in the Implementation to Shorten the Review Cycle for New Investigator R01 Applications. PUBLIC HEALTH RELEVANCE: The proposed research will (1) provide insight into the dynamics of intracellular Cu transporters and yield fundamental knowledge for understanding the causes of Cu transport related diseases, and (2) elucidate how MerR-family regulators control transcription in response to metal ions and contribute to our understanding of metal regulation in humans. The single-molecule methods developed will enable new experiments to address many biological problems and will broadly impact quantitative investigations of health related problems, including studies of biomacromolecules for metal homeostasis.