Supplementary Figure 10: High-resolution ¹⁹F-¹³C TROSY facilitates the detection of minor conformations.

Low-population cross-peaks in the ¹⁹F-¹³C TROSY spectrum of MBP are shown in green circles. These resonances are likely to have originated from rotamers of 3-¹⁹F_13C Tyr due to (1) rotation along the Cγ–Cβ bond or (2) rotation along the phenolic Cζ–O bond, which determines the proximity of the hydroxyl hydrogen atom to the fluorine atom. Similarly, slow flipping of the hydroxyl group around the phenolic Cζ–O bond between an intra-residue O-H-F and an inter-residue H-bonding configuration can produce two sets of resonances of unequal intensity. Such rotamers of 3-¹⁹F Tyr have been previously observed by X-ray crystallography (J. Mol. Biol. 281, 323–339; 1998). In a few instances, for the very strong cross-peaks, we observed a minor conformation that corresponds to the non-TROSY component along the ¹³C dimension; this is marked by a red solid rectangle. The non-TROSY component can be recognized by its ¹⁹F resonance frequency, which is identical to that of its TROSY counterpart with a ¹³C frequency separation of ~240 Hz, corresponding to the ¹J_FC coupling constant.