Figure 1. Strategy for acquiring ¹Hα CEST data on IDPs.

A) The ¹Hα region of a constant-time ¹H-¹³C HSQC spectrum of U-¹³C,¹⁵N labelled CytR^N. Green resonances arise from Gly which do not have a scalar coupled sidechain ¹³Cβ. B) ¹H-¹⁵N HSQC spectrum of CytR^N. C) The coherence transfer pathway in the ¹Hα CEST experiment reported here. CEST is carried out on ¹Hα (black dashed square) and the magnetization is then transferred from ¹Hα(i) through ¹³Cα(i) and ¹³CO(i) to ¹⁵N (i+1) and then to ¹HN(i+1) for detection. Red dashed squares indicate nuclei that are frequency labelled in the experiment. D) Cartoon representation of conformational exchange between ground and excited states of a protein where the alpha proton H₁ comes close (< 5 Å) to H₂ in the ground state. E) The spin-state-selective approach for acquiring proton CEST without interference from dipolar cross-relaxation. (Top) ¹H CEST profile of an alpha proton resonating at 4.5 ppm (major dip), exchanging with a second conformation where the chemical shift of the ¹Hα is 5.5 ppm (minor dip). The ¹Hα is proximal to a second ¹Hα which results in an NOE dip at 3.5 ppm. There is no ¹³C-decoupling during the CEST period; therefore doublets separated by ¹J_HαCα are seen for each dip. (Middle) Spin-state-selective CEST data selecting for H_zC_α (blue) and H_zC_β (red). (Bottom) CEST profile of H_zC_α-H_zC_β =2H_zC_z, where the NOE dip cancels out (green).