Relating dynamic protein interactions of metallochaperones with metal transfer at the single-molecule level

Abstract

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.

Glutathione limits aquacopper(I) to sub-femtomolar concentrations through cooperative assembly of a tetranuclear cluster

“…mechanisms without intermediacy of the free aquated Cu(I) cation. In fact, single molecule FRET studies of the copper metallochaperone Atox1 and its binding partners imply a highly dynamic exchange mechanism involving formation of multiple interaction complexes characterized by short lifetimes (35)(36)(37). Although the transfer of Cu(I) could not be observed directly based on these measurements, the short lifetime of the underlying protein-protein interaction complexes is consistent with rapid Cu(I) transfer through an associative exchange as originally proposed by O'Halloran and co-workers (38).…”

Synthetic fluorescent probes for monovalent copper

“…Once inside the cell, a host of selective metallochaperones are responsible for escorting Cu(I) to various subcellular destinations [5–7]. Although these metallochaperones bind Cu(I) very tightly, typically with dissociation constants in the femto- to attomolar concentration range [8], the metal transfer to the downstream recipient occurs with rapid kinetics [9]. The metallochaperones achieve this by coordinating Cu(I) with a surface-exposed bidentate CXXC motif, which allows for an associative exchange mechanism without transient release of aqueous Cu(I).…”

Single-Molecule Dynamics and Mechanisms of Metalloregulators and Metallochaperones

“…Copyright 2012 American Chemical Society. And reproduced from reference (95) by permission of The Royal Society of Chemistry.…”