A R T I C L E S Otten et al. Figure 4. Representative methyl transverse 1H relaxation dispersion proﬁles for Ile-δ1 and Thr methyl groups for calbindin D9k recorded at 600 MHz. Panels A and C show spurious dispersion proﬁles originating from homonuclear scalar coupling; in panels B and D, the source of this artifact has been removed in the NMR experiment. Figure 5. Methyl transverse 1H relaxation dispersion proﬁle, representative for many downﬁeld resonating Leu-δ1/δ2 methyl groups in the case of strong 13C scalar coupling in the ABX spin system (see text). Panel A shows the spurious dispersion proﬁle obtained for L40-δ1 of calbindin D9k; in panel B, the source of this artifact has been removed in the NMR experiment. and 95% for Ala-R, Leu-γ, Val- , and Ile- , but is signiﬁcantly lower for Ile-γ1 and Thr- positions (66% and 81%, respec- tively).58 Of note, because of the high level of deuteration, the occurrence of a CH2 methylene group is e5% and can, therefore, be neglected. Indeed, in the case of Ile-δ1 and Thr (Figure 4, panels A and C), spurious dispersion proﬁles are observed, because the positions adjacent to the methyl groups are protonated to a signiﬁcant degree, and 1H homonuclear scalar couplings affect the signal intensity due to Hartmann-Hahn mixing, to a degree that depends on the CPMG pulse repetition rate.26 These artifacts can easily be suppressed, by selecting only the magnetization originating from methyl groups for which the neighboring carbon atom is fully deuterated (Figure 1, scheme C). This selection is achieved by dividing the CT period in two parts, separated by an INEPT period (see the Supporting Information for a detailed description). It is obvious from Figure 4 that signiﬁcant artifacts arise from the protonation at the γ1-position of Ile and -position of Thr residues, but also that these can be purged by choosing experimental scheme C (Figure 1). Using this pulse scheme, 17010 J. AM. CHEM. SOC. 9 VOL. 132, NO. 47, 2010 ﬂat lines are obtained for Ile-δ1 and Thr methyl groups as well (Figure 4, panels B and D), with an average rmsd value of 0.22 ( 0.03 s-1 for the two Ile-δ1 and one Thr of calbindin D9k considered here. Of note, as expected, the background R2, eff obtained using scheme C is also lower in this case because the 1H-1H dipolar interaction with the neighboring nuclei is removed.33,34 Finally, it should be noted that in scheme C ∼14 ms is needed to evolve the JCC coupling, which cannot be used at the same time for chemical shift encoding. Therefore, the resolution of the resulting 2D spectra is a factor of 2 lower than for scheme A. Although one could increase the period for chemical shift evolution from 1/(2JCC) to 3/(2JCC) to obtain a higher resolution, at the cost of signal intensity, scheme C is only advantageous for Ile-δ1 and Thr methyl groups, with the proposed labeling. Another type of artifact was observed for several Leu methyl groups, where dispersion proﬁles showed enhanced R2, eff values at low pulsing rates, as shown in Figure 5, panel A. Because the deuteration level of the Leu-γ position is ∼92%, it is unlikely that the spurious proﬁles originate from 1H homonuclear scalar