Microsecond Time Scale Dynamics in Proteins coupling. Furthermore, if 1H-1H coupling would be the source of the spurious proﬁles, then one would expect this to be similar for all Leu residues (and for both the δ1 and the δ2 methyl groups), which is not the case. Moreover, the proﬁles remain identical when scheme C is used to record the CPMG relaxation dispersion experiment (see the Supporting Information for all proﬁles, Figure S1). One potential source of deviation might be strong 13C scalar coupling. It is well-known that the difference between the downﬁeld Leu-δ1/δ2 and the Leu-γ chemical shifts can be rather small (25.7 ( 1.2 versus 26.7 ( 1.3 ppm, respectively),92 and there seems to be a (slight) correlation between this difference and how pronounced the artifacts are. Indeed, “strong coupling effects during X-pulse CPMG experi- ments on heteronuclear ABX spin systems” have been observed before in the case of ribose units of RNA.77 Vallurupalli et al. showed that X-magnetization is lost during the CPMG element in the situation where spins A and B are strongly coupled (in our case: X ) 1HM, A ) Leu-Cδ1/Cδ2, and B ) Leu-Cγ) and demonstrated that these artifacts can be removed by recording a CPMG relaxation experiment on in-phase proton magnetiza- tion, by applying a 13C continuous wave (CW) decoupling ﬁeld during the CPMG element.77 We implemented this approach (Figure 1, scheme B), and in several cases the artifacts indeed disappeared and a ﬂat line was obtained (Figure 5, panel B; rmsd ) 0.28 s-1). However, the minimal condition νCW ) 2 · k · νCPMG limits the maximum νCPMG values to ∼2.5 kHz because the maximum attainable 13C CW ﬁeld strength for a period of 40-80 ms is around 5 kHz. In our case, the maximum value for νCPMG was equal to 1 kHz, and the 13C CW ﬁeld strength determined according to the relation- ship above was between 1.2 and 2.1 kHz, and, therefore, complete decoupling will be achieved only for signals close to the carrier position. The offset between the 13C carrier position and L40-δ1 is small (∼0.8 ppm), and in such cases the strong 13C scalar coupling artifacts will be removed because even for the lowest 13C CW ﬁeld the δ1-carbon is completely decoupled (Figure 5). We observe that especially at lower CPMG pulse rates and depending on the offset not all artifacts are removed and, in fact, sometimes even appear for residue types in situations for which none are present in the rcCPMG version (see the Supporting Information, Figure S1). Therefore, in protein applications the suppression of strong 13C scalar coupling artifacts using the above-mentioned pulse scheme, although possible, is rather inefﬁcient. The use of [U-1H,1-13C]-D-glucose as precursor provides an alternative, and fully effective, procedure for the elimination of strong 13C scalar coupling in Leu residues (see below). The aim of this study was to use all available methyl- containing amino acids to record CPMG relaxation dispersion proﬁles, which requires uniform isotope labeling. Such labeling scheme potentially introduces artifacts, but we have demon- strated above that reliable proﬁles can be recorded for all methyl groups if an appropriate pulse scheme is employed. Alterna- tively, one could speciﬁcally introduce CHD2 isotopomers in an otherwise deuterated background using speciﬁc precursors. Because of such a labeling scheme, CPMG relaxation dispersion proﬁles are guaranteed artifact free, but at the expense of a complete amino acid coverage. In a very recent NMR study of proteasome gating, Kay and co-workers have undertaken the latter approach and also considered the potential of CHD2 methyl groups as probes of millisecond time scale dynamics.65 In their (92) Brath, U.; Akke, M.; Yang, D. W.; Kay, L. E.; Mulder, F. A. A. J. Am. Chem. Soc. 2006, 128, 5718–5727. A R T I C L E S study, CHD2 probes were speciﬁcally introduced for methionine residues, which, due to their signiﬁcant degree of fast (ps) side chain dynamics, can still give sensitive spectra, and for which the residue-speciﬁc assignment was obtained from a combination of NMR spectroscopy and mutagenesis.93 Indeed, when dealing with very high molecular weight systems (the R7 subunit of the 20S core particle from T. acidiphilium weighs 180 kDa), it is necessary to introduce speciﬁc labels without isotope dilution into an otherwise strictly deuterated background. However, for many other smaller (<100 kDa) proteins or their assemblies, a near-complete assignment of methyl groups can be obtained cost-effectively using a partial deuteration scheme, as we have recently demonstrated.58 Importantly, these samples, prepared using protonated glucose in D2O, together with appropriate 1H CPMG relaxation dispersion experiments, can be used to quantify microsecond-millisecond time scale motion for all methyl-containing amino acids. In addition, the same samples can also be used for a comprehensive analysis of fast (ps-ns) time scale dynamics.58 Application to Activation of the Signaling Protein NtrCr. Given the fact that ﬂat 1H CPMG relaxation dispersion proﬁles can be obtained for all methyl groups in the absence of exchange, we now focus our attention on a system that displays functional dynamics on the microsecond time scale. The Nitrogen Regulatory Protein C (NtrC) is a bacterial enhancer- binding protein that activates transcription upon phosphorylation of residue D54 in its N-terminal, regulatory domain (NtrCr). Kern and co-workers have demonstrated that the switch from an inactive to active species involves an extensive change in structure,72 and that the mechanism of activation is best described by a population-shift model, where phosphorylation shifts the pre-existing equilibrium from inactive toward active conformations.73 The exchange rate between these two confor- mations was determined on the basis of 15N CPMG data and the exchange-free transverse relaxation rates obtained from separate measurements and was shown to be 13580 ( 850 s-1.73,75 Moreover, the population of active NtrCr was estimated to be 14 ( 2% in the unphosphorylated form of the protein, based on comparison of chemical shifts of the WT sample with respect to those of the fully active and inactive species.75 Relevant to the current study, NtrCr contains 57 methyl- containing amino acids (15 Ala, 8 Ile, 14 Leu, 5 Met, 5 Thr, and 10 Val residues) supplying 89 potential probes for methyl relaxation dispersion spectroscopy. Initial investigations (data not shown) of methyl group dynamics using 13C T1F measure- ments92 have revealed that leucine residues, in particular, were subject to microsecond time scale dynamics. Ideally, one would like to obtain dispersion proﬁles for all different methyl- containing amino acids from a single experiment, but, as stated earlier, strong 13C scalar coupling in Leu residues remains problematic. As mentioned above, relaxation dispersion proﬁles for Leu methyl groups are of great interest in this system, and, therefore, it would be useful to remove the strong 13C scalar coupling phenomenon using a biochemical approach. As demonstrated earlier by Lundstro¨ m et al.,94 13C enrichment of the methyl groups, while the neighboring carbon atom is of the 12C variety, can be accomplished by the use of [U-1H,1-13C]- D-glucose as sole carbon source. The 13C enrichment of the methyl groups using [U-1H,1-13C]-D-glucose is a factor of 2 (93) Religa, T. L.; Sprangers, R.; Kay, L. E. Science 2010, 328, 98–102. (94) Lundstro¨m, P.; Teilum, K.; Carstensen, T.; Bezsonova, I.; Wiesner, S.; Hansen, D. F.; Religa, T. L.; Akke, M.; Kay, L. E. J. Biomol. NMR 2007, 38, 199–212. J. AM. CHEM. SOC. 9 VOL. 132, NO. 47, 2010 17011