A R T I C L E S Otten et al. Figure 6. Representative methyl transverse 1H relaxation dispersion proﬁles for Ala, Val, Ile-γ2, and Leu methyl groups in NtrCr recorded at 600 (upper panel) and 800 (lower panel) MHz. The solid lines correspond to the best curves, obtained from ﬁtting the experimental data from both ﬁelds simultaneously to the appropriate two-site exchange equation. lower as compared to when U-[1H, 13C]-D-glucose is used as precursor, but is still sufﬁciently high (between 35% for Met and 44% for Ala, Ile-γ2, Leu, and Val methyl groups, respec- tively).94 For Ile-δ1 and Thr methyl groups, the 13C incorpora- tion drops quite dramatically to ∼13%, and in only 25% of the cases are the Ile-δ1 and Thr methyl groups directly attached to a 13C spin94 such that selection against protonation on the neighboring carbon could be performed. The resulting signal- to-noise ratio for these correlations is, therefore, too low, and these were not included in the analysis. It should be emphasized here that using [U-1H,1-13C]-D-glucose we can reliably use 28 Leu methyl groups at the expense of 5 Thr and 8 Ile-δ1 probes. In addition, for Thr and Ile-δ1, one needs to resort to a less sensitive variant (scheme C, Figure 1) to obtain artifact free proﬁles, and this is simultaneously circumvented. In the NtrCr sample prepared using [U-1H,1-13C]-D-glucose as precursor molecule, 76 methyl groups were labeled to a high extent, and 52 assigned and nonoverlapping cross-peaks were observed in the 2D 1H-13C spectra and could be used in the analysis (Figure 2, right panel). Figure 6 shows representative relaxation dispersion proﬁles for some of the methyl groups in NtrCr (see the Supporting Information for all proﬁles, Figure S2). Initially, the experimental data acquired at 600 and 800 MHz were ﬁtted simultaneously on a per-residue basis to the appropriate model without restricting any of the ﬁtting param- eters (see the Supporting Information, Table S2). In total, for 31 residues, no signiﬁcant exchange contribution to the methyl 17012 J. AM. CHEM. SOC. 9 VOL. 132, NO. 47, 2010 proton line width was observed (model 1), as indicated by the ﬂat proﬁles. Furthermore, it was determined from F-statistic tests that 18 residues are best ﬁtted using the fast-exchange equation (model 2), while the remaining three residues fall in the intermediate exchange regime and are, therefore, best ﬁtted by the general two-site exchange (CRJ) equation (model 3). In the case of model 2 (fast-exchange), only the product Φex ) pApBδω2 and τex can be obtained from the analysis. In cases where the exchange is not fast (model 3), it is often possible to separate Φex into its individual components (i.e., populations and chemical shift difference). In our case, however, the three residues that were best ﬁtted to model 3 show very low values for the population of the minor state, pB < 0.3%, and unrealistically large values for the chemical shift difference between the two states (|∆ δ | between 1.8 and 3.2 ppm). Through independent experiments, Gardino et al.75 reliably determined the population pB to 14%, and we therefore used this value for the population of the minor state. By restricting the population to this value, the three residues initially following model 3 are now accurately described by the fast-exchange model. Of note, the ﬁtting parameters of the 18 probes described by model 2 did not change by assuming a value for pB, but the chemical shift difference between the two states can now be calculated according to δω ) Φex p p A B