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

Abstract

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.

Keywords: 1H chemical exchange saturation transfer; Protein dynamics; Rare conformational states; Slow exchange.