A Double-Resonance CEST Experiment To Study Multistate Protein Conformational Exchange: An Application to Protein Folding

Despite the importance of protein dynamics to function, studying exchange between multiple conformational states remains a challenge because sparsely populated states are invisible to conventional techniques. CEST NMR experiments can detect minor states with lifetimes between 5 and 200 ms populated to a level of just similar to 1%. However, CEST often cannot provide the exchange mechanism for processes involving three or more states, leaving the role of the detected minor states unknown. Here a double-resonance CEST experiment to determine the kinetics of multistate exchange is presented. The approach that involves irradiating resonances from two minor states simultaneously is used to study the exchange of T4 lysozyme (T4L) between the dominant native state and two minor states, the unfolded state and a second minor state (B), each populated to only similar to 4%. Regular CEST does not provide the folding mechanism, but double-resonance CEST clearly shows that T4L can fold directly without going through B.