A "Steady-State" Relaxation Dispersion Nuclear Magnetic Resonance Experiment for Studies of Chemical Exchange in Degenerate 1 H Transitions of Methyl Groups.

Degenerate spin-systems consisting of magnetically equivalent nuclear spins, such as a 1 H 3 spin-system in selectively 13 CH 3 -labeled proteins, present considerable challenges for the design of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments to characterize chemical exchange on the micro-to-millisecond time-scale. Several approaches have been previously proposed for the elimination of deleterious artifacts observed in methyl 1 H CPMG relaxation dispersion profiles obtained for ( 13 C) 1 H 3 groups. We describe an alternative, experimentally simple solution and design a "steady-state" methyl 1 H CPMG scheme, where 90° or acute-angle (<90°) 1 H radiofrequency pulses are applied after each CPMG echo in-phase with methyl 1 H magnetization, resulting in the establishment of a "steady-state" for effective rates of magnetization decay. A simple computational procedure for quantitative analysis of the "steady-state" CPMG relaxation dispersion profiles is developed. The "steady-state" CPMG methodology is applied to two protein systems where exchange between major and minor species occurs in different regimes on the chemical shift time-scale.