Probing slow timescale dynamics in proteins using methyl 1H CEST

Abstract

Although 15N- and 13C-based chemical exchange saturation transfer (CEST) experiments have assumed an important role in studies of biomolecular conformational exchange, 1H CEST experiments are only beginning to emerge. We present a methyl-TROSY 1H CEST experiment that eliminates deleterious 1H–1H NOE dips so that CEST profiles can be analyzed robustly to extract methyl proton chemical shifts of rare protein conformers. The utility of the experiment, along with a version that is optimized for 13CHD2 labeled proteins, is established through studies of exchanging protein systems. A comparison between methyl 1H CEST and methyl 1H CPMG approaches is presented to highlight the complementarity of the two experiments.