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

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.