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. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Anderson, Vernon
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Cornell University
Schools of Arts and Sciences
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Chen, Peng; Keller, Aaron M; Joshi, Chandra P et al. (2013) Single-molecule dynamics and mechanisms of metalloregulators and metallochaperones. Biochemistry 52:7170-83
Joshi, Chandra P; Panda, Debashis; Martell, Danya J et al. (2012) Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator. Proc Natl Acad Sci U S A 109:15121-6
Keller, Aaron M; Benítez, Jaime J; Klarin, Derek et al. (2012) Dynamic multibody protein interactions suggest versatile pathways for copper trafficking. J Am Chem Soc 134:8934-43
Benitez, Jaime J; Keller, Aaron M; Huffman, David L et al. (2011) Relating dynamic protein interactions of metallochaperones with metal transfer at the single-molecule level. Faraday Discuss 148:71-82; discussion 97-108
Chen, Peng; Andoy, Nesha May; Benítez, Jaime J et al. (2010) Tackling metal regulation and transport at the single-molecule level. Nat Prod Rep 27:757-67
Benitez, Jaime J; Keller, Aaron M; Chen, Peng (2010) Nanovesicle trapping for studying weak protein interactions by single-molecule FRET. Methods Enzymol 472:41-60
Andoy, Nesha May; Sarkar, Susanta K; Wang, Qi et al. (2009) Single-molecule study of metalloregulator CueR-DNA interactions using engineered Holliday junctions. Biophys J 97:844-52