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

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