Monitoring Macromolecular Motions on Microsecond to Millisecond Time Scales by R_(1ρ)--R_1 Constant Relaxation Time NMR Spectroscopy

摘要

Dynamic processes on microsecond to millisecond (μs--ms) time scales are important for the functions of proteins, including recognition, allostery, and catalysis. Intramolecular motions on μs--ms time scales contribute to nuclear magnetic relaxationthrough adiabatic dephasing of coherent states and are exhibited as conformational exchange phenomena in solution-state NMR spectroscopy. Nuclear magnetic relaxation in the rotating frame (i.e., in the presence of a radiofrequency (rf) field) constitutes a unique source of information on chemical and conformational exchange processes. This communication presents a new rotating frame technique for studying intra- and intermolecular exchange in proteins that overcomes several difficulties associated withexisting spin-lock and spin-echo experiments. First, rotating frame and laboratory frame relaxation rate constants are averaged during a novel constant relaxation time (CRT) period in order to simplify the off-resonance effects normally encountered in spin-lock experiments. Second, an off-resonance spin-lock rf field is used to increase the magnitude of the effective magnetic field in the rotating frame in order to access faster dynamic processes. The off-resonance R_(1ρ)--R_1 CRT nuclear magnetic relaxation experiment allows determination of conformational exchange times at least as short as 25 μs in proteins.