Single-Molecule Study of Dynamics and Mechanisms of Biomacromolecule Interactions

为了维持正常的新陈代谢，生物体使用复杂的机制来介导金属的安全运输 离子的细胞内，并调节基因的表达，对金属诱导的化学应激。的方式 然而，所涉及的生物大分子实现其功能的机制在很大程度上是未知的。我们的长期 目的是了解生物大分子如何共同作用于细胞内金属转运， 通过利用电流和开发新的单分子荧光方法进行金属调节， 作为了解金属代谢相关疾病原因的前提。在这一提议中， 我们集中在（1）人类铜分子伴侣Hah 1如何与铜转运ATP酶Wilson相互作用 疾病蛋白（WDP）的铜运输和（2）如何MerR家族金属调节剂相互作用， 改变DNA的结构以进行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。这里我们将 使用纳米囊泡捕获，使瞬时蛋白质-蛋白质相互作用的单分子FRET研究。 我们的子目标是：1）表征Hah 1和WDP的单金属结合结构域（MBD）如何相互作用， 铜走私2)表征Hah 1和WDP的多结构域构建体如何与铜相互作用 贩卖人口3)表征WDP内分子内结构域间相互作用的动力学及其 偶联到与Hah 1的相互作用，用于铜的运输。 2.定义MerR家族调节因子-DNA相互作用的动力学和机制， 响应性转录调控。我们已经开发了工程化的DNA霍利迪连接（HJ）， 用于蛋白质-DNA相互作用的灵敏且特异的单分子报告物。我们的子目标是：1） 开发，表征和应用工程HJ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)探针 使用工程化HJ的三级调节子-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，以及如何 金属调节剂作用于DNA以调节转录。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，并将广泛影响定量研究 复杂的蛋白质-蛋白质和蛋白质-DNA相互作用网络。 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申请的审核周期。本研究将（1）为深入了解细胞内铜转运蛋白的动力学提供理论依据 并为理解铜转运相关疾病的原因提供基础知识;（2） 阐明MerR家族调节因子如何响应金属离子控制转录，并有助于我们的研究。 了解人类的金属调节。开发的单分子方法将使新的 实验，以解决许多生物学问题，并将广泛影响定量研究， 健康相关的问题，包括金属稳态的生物大分子研究。

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. 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.