A CEST NMR experiment to obtain glycine 1Hα chemical shifts in ‘invisible’ minor states of proteins

Chemical exchange saturation transfer (CEST) experiments are routinely used to study protein conformational exchange between a ‘visible’ major state and ‘invisible’ minor states because they can detect minor states with lifetimes varying from ~ 3 to ~ 100 ms populated to just ~ 0.5%. Consequently several 1H, 15N and 13C CEST experiments have been developed to study exchange and obtain minor state chemical shifts at almost all backbone and sidechain sites in proteins. Conspicuously missing from this extensive set of CEST experiments is a 1H CEST experiment to study exchange at glycine (Gly) 1Hα sites as the existing 1H CEST experiments that have been designed to study dynamics in amide 1H-15N spin systems and methyl 13CH3 groups with three equivalent protons while suppressing 1H-1H NOE induced dips are not suitable for studying exchange in methylene 13CH2 groups with inequivalent protons. Here a Gly 1Hα CEST experiment to obtain the minor state Gly 1Hα chemical shifts is presented. The utility of this experiment is demonstrated on the L99A cavity mutant of T4 Lysozyme (T4L L99A) that undergoes conformational exchange between two compact conformers. The CEST derived minor state Gly 1Hα chemical shifts of T4L L99A are in agreement with those obtained previously using CPMG techniques. The experimental strategy presented here can also be used to obtain methylene proton minor state chemical shifts from protein sidechain and nucleic acid backbone sites.