A new class of CEST experiment based on selecting different magnetization components at the start and end of the CEST...

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.