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.

While semiconductor quantum dots (QDs) have been used successfully in numerous single particle tracking (SPT) studies due to their high photoluminescence efficiency, photostability, and broad palette of emission colors, conventional QDs exhibit fluorescence intermittency or ‘blinking,’ which causes ambiguity in particle trajectory analysis and limits tracking duration. Here, non-blinking ‘giant’ quantum dots (gQDs) are exploited to study IgE-FcεRI receptor dynamics in live cells using a confocal-based 3D SPT microscope. There is a 7-fold increase in the probability of observing IgE-FcεRI for longer than 1 min using the gQDs compared to commercially available QDs. A time-gated photon-pair correlation analysis is implemented to verify that selected SPT trajectories are definitively from individual gQDs and not aggregates. The increase in tracking duration for the gQDs allows the observation of multiple changes in diffusion rates of individual IgE-FcεRI receptors occurring on long (>1 min) time scales, which are quantified using a time-dependent diffusion coefficient and hidden Markov modeling. Non-blinking gQDs should become an important tool in future live cell 2D and 3D SPT studies, especially in cases where changes in cellular dynamics are occurring on the time scale of several minutes.

The distributions of the individual waiting times in Figure 2D were analyzed incorrectly. In the protein interaction scheme ( Figure 2C), each of the E FRET states (E 0 , E 1 , and E 2 ) branches directly to two other states. The decay constant from each of the six waiting time distributions (i.e., τ 0f1 , τ 0f2 , τ 1f0 , τ 1f2 , τ 2f0 , and τ 2f1 ) does not directly correspond to a particular kinetic constant in the interaction scheme, but instead is the sum of the rate constants of the two kinetic processes that branch from the same state (i.e., E 0 , E 1 , or E 2 ). The individual rate constants can subsequently be determined using the ratios of the number of transition events for each kinetic process. The relations between the decay constants of the waiting time distributions and the rate constants are given in the revised Figure 2D below; their derivations are in the additional Supporting Information. The decay constants of the τ 0f1 and τ 0f2 distributions should be the same, as should those of the τ 1f0 and τ 1f2 distributions and those of the τ 2f0 and τ 2f1 distributions. By omitting the first bin in each waiting time distribution, which is often inaccurate due to limited time resolution, all six waiting time distributions can be fitted consistently with single-exponential decay functions ( Figure 2D). The results give k 1 ) (1., and k -3 ) 0.7 ( 0.1 s -1. From these rate constants, we can also obtain the dissociation constants for the two interaction complexes with K 1 ) 5.6 ( 0.6 µM and K 2 ) 9 ( 1 µM (see also revised Figure S6 in the Supporting Information). Except for the quantitative values of the kinetic parameters listed here, this correction does not affect any other conclusions in our study. We thank Taekjip Ha for alerting us to the error. Supporting Information Available:Derivation of waiting time distribution for branching processes and revised Figure S6. This material is available free of charge via the Internet at http://pubs.acs.org.

A new compact and multifunctional hybrid semiconductor–metal nanostructure is elucidated and demonstrated for real-time optical imaging, photothermal heating, and in situ thermometry.

SummaryMetallochaperones undertake specific interactions with their target proteins to deliver metal ions inside cells. Understanding how these protein interactions are coupled with the underlying metal transfer process is important, but challenging because they are weak and dynamic. Here we use a nanovesicle trapping scheme to enable single-molecule FRET measurements of the weak, dynamic interactions between the copper chaperone Hah1 and the fourth metal binding domain (MBD4) of WDP. By monitoring the behaviors of single interacting pairs, we visualize their interactions in real time in both the absence and the presence of various equivalents of Cu 1+ . Regardless of the proteins' metallation state, we observe multiple, interconverting interaction complexes between Hah1 and MBD4. Within our experimental limit, the overall interaction geometries of these complexes appear invariable, but their stabilities are dependent on the proteins' metallation state. In apo-holo Hah1-MBD4 interactions, the complexes are stabilized relative to that observed in the apo-apo interactions. This stabilization is undiscernible when Hah1's Cu 1+ -binding is eliminated or when both proteins have Cu 1+ loaded. The nature of this Cu 1+ -induced complex stabilization and of the interaction complexes are discussed. These Cu 1+ -induced effects on the Hah1-MBD4 interactions provide a step toward understanding how the dynamic protein interactions of copper chaperones are coupled with their metal transfer function.

Single-molecule fluorescence resonance energy transfer (smFRET) remains a widely utilized and powerful tool for quantifying heterogeneous interactions and conformational dynamics of biomolecules. However, traditional smFRET experiments either are limited to short observation times (typically less than 1 ms) in the case of "burst" confocal measurements or require surface immobilization which usually has a temporal resolution limited by the camera framing rate. We developed a smFRET 3D tracking microscope that is capable of observing single particles for extended periods of time with high temporal resolution. The confocal tracking microscope utilizes closed-loop feedback to follow the particle in solution by recentering it within two overlapping tetrahedral detection elements, corresponding to donor and acceptor channels. We demonstrated the microscope's multicolor tracking capability via random walk simulations and experimental tracking of 200 nm fluorescent beads in water with a range of apparent smFRET efficiency values, 0.45-0.69. We also demonstrated the microscope's capability to track and quantify double-stranded DNA undergoing intramolecular smFRET in a viscous glycerol solution. In future experiments, the smFRET 3D tracking system will be used to study protein conformational dynamics while diffusing in solution and native biological environments with high temporal resolution.