Tackling metal regulation and transport at the single-molecule level

To maintain normal metal metabolism, organisms utilize dynamic cooperation of many biomacromolecules for regulating metal ion concentrations and bioavailability. How these biomacromolecules work together to achieve their functions is largely unclear. For example, how do metalloregulators and DNA interact dynamically to control gene expression to maintain healthy cellular metal level? And how do metal transporters collaborate dynamically to deliver metal ions? Here we review recent advances in studying the dynamic interactions of macromolecular machineries for metal regulation and transport at the single-molecule level: (1) The development of engineered DNA Holliday junctions as single-molecule reporters for metalloregulator-DNA interactions, focusing onMerR-family regulators. And (2) The development of nanovesicle trapping coupled with single molecule fluorescence resonance energy transfer (smFRET) for studying weak, transient interactions between the copper chaperone Hah1 and the Wilson disease protein. We describe the methodologies,the information content of the single-molecule results, and the insights into the biological functions of the involved biomacromolecules for metal regulation and transport. We also discuss remaining challenges from our perspective.