Metals are essential nutrients that can also be toxic. Safe trafficking of metal ions is necessary inside cells, and specific metal transport pathways exist to deliver them to their destinations. 1,2 In human cells, the copper chaperone Hah1 and the Wilson disease protein (WDP) constitute a copper transport pathway-Hah1 is a single-domain cytoplasmic protein; WDP is a multidomain protein anchored on organelle membranes and has a cytosolic N-terminal region consisting of six homologous metal-binding domains (MBDs). All WDP MBDs and Hah1 contain a conserved CXXC motif that binds Cu 1+ , and Cu 1+ is transferred from Hah1 to a WDP MBD via direct and specific 3,4 Although the MBDs of WDP have different functional roles, 4,5 all of them, as well as Hah1, have similar Cu 1+ binding affinities. 3 This similarity indicates that the Hah1 to WDP Cu 1+ transfer is under kinetic control mediated by Hah1-WDP interactions, and that the functional differences among WDP MBDs are not defined by their Cu 1+ binding abilities but may be related to how each MBD interacts with Hah1. Very limited quantitative information is available, however, on the Hah1-WDP interaction dynamics. This is partly because the Hah1-WDP interactions are transient, and transient interactions are difficult to quantify in ensembleaveraged experiments.Here we report using nanovesicle trapping and single-molecule fluorescence resonance energy transfer (smFRET) measurements to probe the transient interactions between Hah1 and the fourth MBD (MBD4) of WDP in real time. We chose MBD4 as a representative WDP MBD because it is known to interact with Hah1 directly for Cu 1+ transfer. 4,6 Quantification of Hah1-MBD4 interaction dynamics will help understand how Hah1 and the full length WDP interact for Cu 1+ transfer.A primary obstacle in single-molecule experiments to probe transient protein interactions is the low concentrations (10 −12 -10 −9 M) commonly used to spatially separate molecules for detection, which limits the experiments to strong protein interactions. Weak protein interactions, including Hah1-WDP interactions, need to be studied at higher concentrations. Nonspecific protein-glass surface interactions during molecule immobilization present another challenge and must be minimized.To overcome these challenges, we adapted a nanovesicle trapping strategy (Figure 1), which was used to study protein and RNA folding and DNA-protein interactions at the singlemolecule level. 7 We trapped the two interacting molecules in a 100 nm diameter lipid vesicle. Because of the confined volume (∼5 × 10 −19 L), the effective concentration is ∼3 μM for each protein inside. Low concentrations of vesicles are then immobilized on a lipid bilayer or polymer-coated glass surface so protein-glass interactions are eliminated.To report Hah1-MBD4 interactions by smFRET, we introduced a C-terminal cysteine in both Hah1 and MBD4 and labeled this cysteine of Hah1 with Cy5 and that of MBD4 with Cy3. Cy3-Cy5 form a FRET pair with a Förster radius of ∼6 nm. The cysteines in the CXXC mot...

As part of intracellular copper trafficking pathways, the human copper chaperone Hah1 delivers We proposed 2-body and 3-body interaction models based on previously known structures of homologous proteins to describe both intermolecular Hah1-MBD and intramolecular MBD-MBD interactions. These interaction models and our smFRET results were then used to formulate and quantify a comprehensive Hah1-MBD34 mechanism. The enhanced interaction stability of Hah1 with the multi-MBD system, the dynamic intramolecular MBDMBD interactions, and the ability of Hah1 to interact with multiple MBDs simultaneously suggest an efficient and versatile mechanism for the Hah1-to-WDP pathway to transport Cu Last, but not least, I would like to thank my wife, Dixie, and daughter, Lilly. Their love and support was critical, and I will forever be in debt to their future vitality and happiness.

Protein–protein interactions are fundamental biological processes. While strong protein interactions are amenable to many characterization techniques including crystallography, weak protein interactions are challenging to study due to their dynamic nature. Single-molecule FRET can monitor dynamic protein interactions in real time, but are generally limited to strong interacting pairs because of the low concentrations needed for single-molecule detection. Here we describe a nanovesicle trapping approach to enable single-molecule FRET study of weak protein interactions at high effective concentrations. We describe the experimental procedures, summarize the application in studying the weak interactions between intracellular copper transporters, and detail the single-molecule kinetic analysis of bimolecular interactions involving three states. Both the experimental approach and the theoretical analysis are generally applicable for studying many other biological processes at the single-molecule level.