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The function of many proteins involves equilibria between conformational substates, and to elucidate mechanisms of function it is essential to have experimental tools to detect the presence of conformational substates and to determine the time scale of exchange between them. Site-directed spin labeling (SDSL) has the potential to serve this purpose. In proteins containing a nitroxide side chain (R1), multicomponent electron paramagnetic resonance (EPR) spectra can arise either from equilibria involving different conformational substates or rotamers of R1. To employ SDSL to uniquely identify conformational equilibria, it is thus essential to distinguish between these origins of multicomponent spectra. Here we show that this is possible based on the time scale for exchange of the nitroxide between distinct environments that give rise to multicomponent EPR spectra; rotamer exchange for R1 lies in the ≈0.1–1 μs range, while conformational exchange is at least an order of magnitude slower. The time scales of exchange events are determined by saturation recovery EPR, and in favorable cases, the exchange rate constants between substates with lifetimes of approximately 1–70 μs can be estimated by the approach.

Resolving Conformational and Rotameric Exchange in Spin-Labeled Proteins Using Saturation Recovery EPR