Capturing protein dynamics across timescales with site-directed spin labeling electron paramagnetic resonance spectroscopy

In the current age of protein structure prediction and determination, resolving the time dependence of structural transitions represents an exciting frontier. Time-resolved biophysical techniques possess the capability to directly observe dynamic structural changes of biomolecules in real time. Here, we review applications of site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy that cover a broad range of protein dynamics, from backbone fluctuations on the ps–ns timescale to protein complex assembly formation on the ms–s timescale. Recent developments in SDSL EPR methods allow for direct investigation of protein conformational exchange kinetics on the important μs–ms timescale, providing the time axis for structural transitions needed to define molecular mechanisms of complex biological phenomena.