Abstract
Biomolecular dynamics in the microsecond-to-millisecond (µs-ms) timescale are linked to various biological functions, such as enzyme catalysis, allosteric regulation, and ligand recognition. In solution state NMR, Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments are commonly used to probe µs-ms timescale motions, providing detailed kinetic, thermodynamic, and mechanistic information at the atomic level. For investigating conformational dynamics in high-molecular-weight biomolecules, methyl groups serve as ideal probes due to their favorable relaxation properties, and (13)C CPMG relaxation dispersion is widely employed for characterizing dynamics in selectively (13)CH(3)-labeled samples. However, conventional schemes that apply CPMG pulses with constant phase are susceptible to artifacts arising from off-resonance effects, radiofrequency (RF) field inhomogeneity and pulse imperfections. In this work we present an optimized(13)C single-quantum (SQ) CPMG experiment incorporating the [0013]-phase cycling scheme, and demonstrate its enhanced robustness against various adverse effects. Moreover, the optimized pulse scheme enables finer sampling of CPMG pulsing frequencies and is suited for studying systems with variable J(CH) scalar coupling constants, thereby facilitating comprehensive characterization of µs-ms timescale dynamics of biomolecules with increased precision.