Measurement of Protein Dynamics from Site Directed Cu(II) Labeling

This review describes the use of Electron Paramagnetic Resonance (EPR) to measure residue specific dynamics in proteins with a specific focus on Cu(II)-based spin labels. First, we outline approaches used to measure protein motion by nitroxide-based spin labels. Here, we describe conceptual details and outline challenges that limit the use of nitroxide spin labels to solvent-exposed alpha-helical sites. The bulk of this review showcases the use of newly developed Cu(II)-based protein labels. In this approach, the strategic mutation of native residues on a protein to generate two neighboring Histidine residues (i.e., the dHis motif) is exploited to enable a rigid site-selective binding of a Cu(II) complex. The chelation of the Cu(II) complex to dHis directly anchors the Cu(II) spin label to the protein backbone. The improvement in rigidity expands both the spin-labeling toolkit as well as the resolution of many EPR measurements. We describe how EPR measurements of the Cu(II) label directly reflect backbone motion and fluctuations. The EPR are complemented by Molecular Dynamics simulations. Finally, the dHis motif provides access to the measurement of site-specific dynamics at both alpha-helices and beta-sheets. The review outlines the limitations of the dHis method and provides an outlook for future developments.

Automated summary

This review focuses on the use of Electron Paramagnetic Resonance (EPR) to measure residue-specific dynamics in proteins, with a specific emphasis on Cu(II)-based spin labels. It discusses the limitations of nitroxide-based spin labels and showcases the use of new Cu(II)-based protein labels, which improve rigidity and expand the spin-labeling toolkit. The review also describes how EPR measurements of the Cu(II) label reflect protein backbone motion and fluctuations, and provides insights into the measurement of site-specific dynamics at both alpha-helices and beta-sheets using the dHis motif. The review concludes by outlining the limitations of the dHis method and suggesting future developments.