Single-Molecule Study of Dynamics and Mechanisms of Biomacromolecule Interactions

描述（由申请人提供）：为了维持正常的代谢，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，并调节基因的表达，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运输和金属调节，作为了解金属代谢相关疾病原因的先决条件。在本研究中，我们主要关注(1)人类铜伴侣蛋白ha1如何与铜转运ATPase Wilson病蛋白（WDP）相互作用以进行铜转运；(2)mer家族金属调节因子如何与DNA相互作用并改变DNA结构以进行金属响应性转录调控。我们的具体目标是：1。定义Hah1-WDP相互作用对铜贩运的动力学和机制。在这里，我们将使用纳米囊泡捕获来实现瞬时蛋白质-蛋白质相互作用的单分子FRET研究。我们的子目标是：1)表征h_1和WDP的单金属结合域（MBDs）如何相互作用以运输铜。2)表征ha1和WDP多结构域构建体在铜转运中的相互作用。3)表征WDP分子内结构域间相互作用的动力学特征，以及它们与ha1相互作用对铜运输的耦合。2. 定义金属响应性转录调控的mrr家族调控因子- dna相互作用的动力学和机制。我们已经开发了工程DNA Holliday连接（HJs）作为蛋白质-DNA相互作用的敏感和特异性单分子报告。我们在这里的子目标是：1)开发，表征和应用工程hj来报告mcr家族调控因子- dna相互作用。2)利用工程HJs探针mir家族调控子施加的DNA解绕进行转录激活。3)利用工程HJs探测三级调控因子- rna聚合酶- dna相互作用。这些研究将深入了解金属转运蛋白如何协同递送金属离子，以及金属调节因子如何作用于DNA来调节转录。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，并将对复杂蛋白质-蛋白质和蛋白质- dna相互作用网络的定量研究产生广泛影响。PI符合NIH对新研究者的定义，并有资格参与缩短新研究者R01申请审查周期的实施。公共卫生相关性：拟议的研究将(1)提供对细胞内铜转运体动力学的见解，并为理解铜转运相关疾病的原因提供基础知识；(2)阐明mcr家族调节因子如何控制金属离子响应的转录，并有助于我们对人类金属调控的理解。所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，包括金属体内平衡的生物大分子研究。

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.