Probing Transient Copper Chaperone−Wilson Disease Protein Interactions at the Single-Molecule Level with Nanovesicle Trapping

Abstract

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...

Single-molecule approaches to characterizing kinetics of biomolecular interactions

“…Following a similar reasoning, researchers have been able to capture fluorescently labeled binding partners in small (100-200 nm), surface-tethered lipid nanovesicles and study their interactions through single-molecule FRET measurements (Figure 2b) [51][52][53]54 ]. The volume of a 100-nm vesicle is two orders of magnitude smaller than that of a diffraction-limited volume and thus allows proteins to be visualized at the single-molecule level at much higher effective concentrations than hitherto possible.…”

Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles

“…A very attractive alternative is to restrict individual molecules within small, optically transparent containers. Examples include lipid vesicles (20)(21)(22)(23)(24), selfassembled polymer nanocapsules (25,26), protein shells of viral capsids (27), and femtoliter chambers chemically etched into the surface of glass optical fibers (28,29).…”

Single-molecule FRET of protein–nucleic acid and protein–protein complexes: Surface passivation and immobilization

“…9a) (58). Benitez et al were able to observe two transiently bound states with different FRET ratios (E 1 ~ 0.5, E 2 ~ 0.9) that can interconvert and quantify all rate constants for the binding scheme (Fig.…”