Single-Molecule Study of Dynamics and Mechanisms of Biomacromolecule Interactions

描述(申请人提供)：为了维持正常的新陈代谢，生物体使用复杂的机制来调节金属离子在细胞内的安全运输，并调节基因的表达，以对抗金属诱导的化学压力。然而，相关的生物大分子如何实现其功能在很大程度上是未知的。我们的长期目标是了解生物大分子如何通过利用电流和开发新的单分子荧光方法来共同作用于细胞内的金属运输和金属调控，作为了解金属代谢相关疾病原因的先决条件。在这个建议中，我们关注(1)人类铜伴侣蛋白Hah1如何与铜转运ATPase Wilson病蛋白(WDP)相互作用进行铜运输，以及(2)MERR家族金属调控因子如何与DNA相互作用并改变其结构，以实现金属响应的转录调控。我们的具体目标是：1.明确HAH1-WDP相互作用在铜交易中的动力学和机制。在这里，我们将使用纳米小室捕获来实现对瞬时蛋白质-蛋白质相互作用的单分子FRET研究。我们的次级目标是：1)研究HAH1和WDP的单金属结合域(MBD)如何相互作用于铜的运输。2)表征HAH1和WDP的多域结构如何相互作用于铜的贩运。3)表征WDP内部分子间相互作用的动力学及其与HAH1相互作用的耦合作用，以实现铜的迁移。2.明确MERR家族调节因子-DNA相互作用对金属反应转录调控的动力学和机制。我们已经开发出工程DNA Holliday连接(HJS)，作为蛋白质-DNA相互作用的灵敏和特异的单分子报告程序。我们的次级目标是：1)开发、鉴定和应用工程HJS来报告Merr家族调节子-DNA的相互作用。2)利用工程HJS探测Merr家族调控因子诱导的DNA解离以进行转录激活。3)利用工程HJS探测三级调节子-RNA聚合酶-DNA的相互作用。这些研究将提供对金属转运体如何协作传递金属离子以及金属调节剂如何作用于DNA以调节转录的洞察。研究中开发的单分子方法将使生物医学研究的新实验成为可能，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对新调查员的定义，并有资格参与实施，以缩短新调查员R01申请的审查周期。与公共健康相关：拟议的研究将(1)提供对细胞内铜转运蛋白动态的洞察，并为理解铜运输相关疾病的原因提供基础知识，以及(2)阐明Merr家族调控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控。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，并将广泛影响与健康相关的问题的定量研究，包括金属动态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.