PELDOR to the Metal: Cu(II)-Based Labels Put a New Spin on Distance Measurements

Eighty years ago, the advent of electron paramagnetic resonance (EPR) revolutionized our ability to observe the physical world of unpaired electron spins. The inception of EPR spawned multiple scientific areas with a focus on discerning the roles of paramagnetic metals and organic radicals in an array of processes and materials. More recently, the emergence of site-directed spin labeling combined with distance measurement technology and molecular modeling has harnessed the power of EPR, to ‘watch proteins move’. Spin labels have enabled the measurement of distance constraints and site-specific dynamics in biomolecules to provide rich details of structure and structural changes that are tightly linked to biological function. Historically, nitroxide radicals are the most common spin labels. However, decades of method development and technological innovation have created a plethora of spin label types to extend the reach of EPR throughout the realm of biophysics. In this review we overview recent developments that improve the sensitivity of distance measurements using Cu(II) labels. These achievements over the last three years promise advancements in the ability of EPR to measure structural and dynamical constraints beyond what is possible using common spin labels. First, we briefly discuss pulsed and continuous-wave EPR techniques that discern the coordination of Cu(II) to monitor spin-labeling efficiency and binding in biological environments. Next, we outline the  bottlenecks that impact sensitivity  in pulsed dipolar spectroscopy and the strategic steps taken to remove these bottlenecks to collect distance measurements in hours. More precisely, we focus on the fast-spin phase memory relaxation time, the broad EPR spectrum due to anisotropy, and orientational selectivity effects inherent to Cu(II). Finally, we showcase the versatile application of Cu(II) spin labels in biological systems and the advantages of Cu(II) in pulsed dipolar spectroscopy to access nanomolar protein concentrations.