Probing slowly exchanging protein systems via 13Cα-CEST: monitoring folding of the Im7 protein

A ¹³C^α chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an ‘observable’ ground state and one or more ‘invisible’ excited states. Here a labeling strategy whereby 2-¹³C]-glucose is the sole carbon source is exploited, producing proteins with ¹³C at the C^α position, while the majority of residues remain unlabeled at CO or C^β. The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state ¹³C^α chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.