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 N-15- and C-13-CEST have been the approaches of choice, the development of spin state selective H-1-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: significantly increases the utility of H-1-based experiments. Pulse schemes have been described previously for studies of highly deuterated proteins. We present here longitudinal-relaxation optimized amide H-1-CEST experiments for probing chemical exchange in protonated proteins. Applications involving a pair of proteins are presented establishing that accurate H-1 chemical shifts of sparsely populated conformers can be obtained from simple analyses of H-1-CEST profiles. A discussion of the inherent differences between N-15-/C-13- and H-1-CEST experiments is presented, leading to an optimal strategy for recording H-1-CEST experiments.