15N CPMG relaxation dispersion experiments for investigating μs-ms timescale dynamics of biomolecules with reduced sample heating effects.

Biomolecular dynamics on the microsecond-to-millisecond (μs-ms) timescale are intimately linked to a wide range of biological functions. In solution-state NMR, Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments are widely employed to probe such motions, providing detailed kinetic, thermodynamic, and mechanistic insights at the atomic level. For studies of protein conformational dynamics, 15N spin probes in backbone amide groups are most commonly used due to the simplicity of the 15N spin system and the ease of sample preparation. Among various 15N CPMG methodologies, in-phase CPMG (ip-CPMG) scheme generally outperforms relaxation-compensated CPMG (rc-CPMG) approach owing to the more favorable relaxation property. However, this experiment typically requires strong radiofrequency (RF) field for 1H decoupling (≥15 kHz), which can be problematic for studying high-salt samples due to increased pulse width on cryogenic probes. Moreover, the strong RF field can induce substantial sample heating effects, potentially perturbing μs-ms timescale dynamics under investigation. In this work we present a new 15N ip-CPMG scheme that enables efficient 1H decoupling with substantially lower RF field strength (∼6 kHz). In addition, an optimized 15N rc-CPMG scheme that yields comparable relaxation dispersion profiles and exchange parameters is also introduced. These new approaches significantly reduce sample heating effects associated with 1H decoupling and are therefore better suited for studying protein samples under high-salt conditions that mimic physiological ionic strengths, enabling more reliable and comprehensive characterization of μs-ms timescale biomolecular dynamics.