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.