Figure 1. NMR spectrum acquired on a 0.5 mM sample of , ‐enriched ubiquitin with a 16.4 T Bruker Avance spectrometer equipped with a cryogenically cooled probehead optimized for detection (cryo‐TXO). The spectrum was recorded with 64 scans using a standard pulse sequence with and decoupling during the acquisition. In the insets, the expansion of the signals at 170.8 and 67.7 ppm is reported to show the extent of the signal splitting due to the C ^α coupling for the C′ signal (around 54 Hz) as well as the C′ and C ^β couplings for the C ^α signal (around 54 and 35 Hz). The ribbon diagram of ubiquitin is also shown.

Figure 2. The fine structure of carbonyls (C′) and α‐carbons (C ^α) in uniformly ‐enriched proteins is schematically indicated, along with the possible experimental approaches to perform carbonyl homonuclear decoupling (band‐selective homonuclear decoupling and two examples of virtual decoupling methods – the IPAP and S ³E variants). In the latter case, the one‐dimensional versions of the experiment to acquire the two independent components of the signal are also shown along with a schematic representation of the linear combinations (and subsequent manipulations) necessary to remove the signal splittings.

Figure 3. Scheme of the correlations that can be detected through the suite of ‐detected exclusively heteronuclear NMR experiments based on carbonyl direct detection. The scalar couplings actively exploited in these experiments are also indicated schematically in the top panel.

Figure 4. Examples of different approaches to acquire the CON and CACO experiments. (a) Two‐dimensional CON spectrum acquired on ubiquitin. The trace of the cross‐peak indicated by the arrow (the correlation for C′59–N60) recorded with different pulse schemes and different relaxation delays (d ₁): (b) start (d ₁= 2.5 s), (c) start (d ₁= 1.5 s), and (d–j) H ^α‐flip acquired with different relaxation delays d ₁ (1.5, 1.2, 0.9, 0.7, 0.5, 0.35, and 0.2 s). (k and l) Two‐dimensional CACO map recorded on ubiquitin with the (H‐flip)CACO experiment without aliasing (around 3 min, k) and with aliasing (around 1 min, l) are shown.