Triple resonance-based 13Cα and 13Cβ CEST experiments for studies of ms timescale dynamics in proteins

A pair of triple resonance based CEST pulse schemes are presented for measuring C-13(alpha) and C-13(beta) chemical shifts of sparsely populated and transiently formed conformers that are invisible to traditional NMR experiments. CEST profiles containing dips at resonance positions of C-13(alpha) or C-13(beta) spins of major (ground) and minor (excited) conformers are obtained in a pseudo 3rd dimension that is generated by quantifying modulations of cross peaks in N-15, H-1(N) correlation spectra. An application to the folding reaction of a G48A mutant of the Fyn SH3 domain is presented, illustrating and validating the methodology.