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

A¹³C(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-(13)C]-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 (13)C(α) chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.