A R T I C L E S suppressing homonuclear scalar couplings26,34,49 and 1H-1H dipolar cross relaxation.50,51 Meanwhile, some methods have emerged that use protons in the backbone as probes of protein dynamics. More speciﬁcally, 1H CPMG relaxation dispersion experiments have been described for amide30,31,34 and alpha35,36 proton nuclei using (per-)deuterated NMR samples and an appropriate pulse scheme. It would be of great interest to extend the 1H CPMG relaxation dispersion methodology to side chain atoms, and methyl groups in particular. Not only are these moieties abundant in proteins, thereby supplying a lot of potential probes to measure dynamics, they are also often located at interesting positions (e.g., in the hydrophobic core and at the interface of complexes)52 and possess favorable NMR properties. The rapid 3-fold jumps about the methyl symmetry axis scales the 13C relaxation rate53 by 1/9, and the fact that the TROSY principle54 can be employed55-57 assures that narrow NMR resonances are observed, even in the case of high molecular weight systems. In addition, high-quality data can be recorded over a large temperature interval, which is important for determining the thermodynamic parameters of the exchange process. Temper- ature-dependent studies using 1HN or 15N CPMG relaxation dispersion measurements are seriously hampered by spin decorrelation effects (e.g., exchange with the solvent), leading to loss of signal in 1H-15N (TROSY-)HSQC experiments. Because methyl protons are nonlabile, they are not affected by this phenomenon, such that high-quality 1H-13C correlation maps can also be recorded at elevated temperature and pH. Here, we describe an approach to record 1H CPMG relaxation dispersion data on methyl groups, selecting for the CHD2 isotopomer. The production of protein samples by bacterial expression using U-[1H, 13C]-D-glucose and ∼100% D2O ensures the isotopic enrichment of all possible methyl groups together with high deuteration levels in the side chain.58-60 Because residual protonation at positions adjacent to the methyl group in the side chain will defeat our strategy, we ﬁrst validated the proposed methodology on calbindin D9k, which has been studied in great detail using NMR spectroscopy and where slow motions are absent.61-64 We are able to demonstrate that 1H CPMG (49) Korzhnev, D. M.; Mittermaier, A. K.; Kay, L. E. J. Biomol. NMR 2005, 31, 337–342. (50) Vold, R. L.; Chan, S. O. J. Chem. Phys. 1972, 56, 28–31. (51) Ishima, R.; Louis, J. M.; Torchia, D. A. J. Magn. Reson. 1999, 137, 289–292. (52) Janin, J.; Miller, S.; Chothia, C. J. Mol. Biol. 1988, 204, 155–164. (53) Kay, L. E.; Bull, T. E.; Nicholson, L. K.; Griesinger, C.; Schwalbe, H.; Bax, A.; Torchia, D. A. J. Magn. Reson. 1992, 100, 538–558. 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Chem. 2003, 41, 853–865. (64) Paquin, R.; Ferrage, F.; Mulder, F. A. A.; Akke, M.; Bodenhausen, G. J. Am. Chem. Soc. 2008, 130, 15805–15807. 17006 J. AM. CHEM. SOC. 9 VOL. 132, NO. 47, 2010 Otten et al. relaxation dispersion proﬁles can be faithfully acquired using a standard CPMG pulse scheme for Ala, Val-γ1/γ2, Leu-δ1/δ2, Ile-γ2, and Met methyl groups, because of the high deuteration level (90-95%) in these amino acid side chains. Spurious dispersion proﬁles were observed in a few cases, and either resulted from signiﬁcant protonation on neighboring carbon atoms (Ile-δ1 and Thr methyl groups) or strong 13C scalar coupling effects (Leu). A straightforward modiﬁcation of the experiment is presented to remove artiﬁcial dispersion proﬁles originating from residual protonation, and solutions are described that resolve strong 13C scalar coupling artifacts, by using a biochemical or NMR approach. The approach we have taken differs in an important way from alternative methods to measure slow (µ s-ms) motions in proteins by 1H relaxation dispersion NMR, as the previously described methods rely on the use of speciﬁcally methyl- protonated precursors on a fully deuterated background.43,65 Although this is key for truly high (>100 kDa) molecular weight systems, speciﬁc labeling considerably limits the number of available probes. Depending on the type of precursor, samples labeled with I(δ1)LV,66,67 Ala,68,69 or Met70 contain approx- imately 60%, 17%, or 3% of methyl groups in proteins, respectively. The utility of our approach is illustrated with an application to the N-terminal receiver domain of the Nitrogen Regulatory Protein C (NtrCr).71-75 NtrC is a transcriptional activator, and the switch from an inactive to active conformation, triggered by phosphorylation of residue D54 in the N-terminal domain (NtrCr), is accompanied by a considerable structural change. 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