Chemical exchange saturation transfer (CEST) experiments are becoming increasingly popular for investigating biomolecular exchange dynamics with rates on the order of approximately 50-500 s-1 and a rich toolkit of different methods has emerged over the past few years. Typically, experiments are based on the evolution of longitudinal magnetization, or in some cases two-spin order, during a fixed CEST relaxation delay, with the same class of magnetization prepared at the start and selected at end of the CEST period. Here we present a pair of TROSY-based pulse schemes for recording amide and methyl 1H CEST profiles where longitudinal magnetization at the start evolves to produce two-spin order that is then selected at the completion of the CEST element. This selection process subtracts out contributions from 1H-1H cross-relaxation on the fly that would otherwise complicate analysis of the data. It also obviates the need to record spin-state selective CEST profiles as an alternative to eliminating NOE effects, leading to significant improvements in sensitivity. The utility of the approach is demonstrated on a sample of a cavity mutant of T4 lysozyme that undergoes chemical exchange between conformations where the cavity is free and occupied.
Keywords: 1H CEST; Chemical exchange; Longitudinal order; NOE dips; TROSY.