Longitudinal relaxation optimized amide 1H-CEST experiments for studying slow chemical exchange processes in fully protonated proteins.

Chemical Exchange Saturation Transfer (CEST) experiments are increasingly used to study slow timescale exchange processes in biomolecules. Although 15N- and 13C-CEST have been the approaches of choice, the development of spin state selective 1H-CEST pulse sequences that separate the effects of chemical and dipolar exchange [T. Yuwen, A. Sekhar and L. E. Kay, Angew Chem Int Ed Engl 2016 doi: 10.1002/anie.201610759 (Yuwen et al. 2017)] significantly increases the utility of 1H-based experiments. Pulse schemes have been described previously for studies of highly deuterated proteins. We present here longitudinal-relaxation optimized amide 1H-CEST experiments for probing chemical exchange in protonated proteins. Applications involving a pair of proteins are presented establishing that accurate 1H chemical shifts of sparsely populated conformers can be obtained from simple analyses of 1H-CEST profiles. A discussion of the inherent differences between 15N-/13C- and 1H-CEST experiments is presented, leading to an optimal strategy for recording 1H-CEST experiments.