A R T I C L E S Figure 7. Cartoon representation of unphosphorylated, inactive state of NtrCr. The spheres represent the carbon atoms of all available methyl group probes, and these are color-coded from red to yellow according to the value of ∆ δ. The structural representation was generated in PyMol.95 process on a 15 000 per second time scale map to the interface between secondary structure elements that were previously identiﬁed to comprise the molecular switch. In conclusion, we have shown how reliable 1H CPMG relaxation dispersion proﬁles up to 6 kHz ﬁeld can be obtained for all methyl groups in protein samples grown on U-[1H, 13C]- D-glucose in D2O. A relaxation compensated 1H CPMG experi- ment is presented for CHD2 methyl groups and yields very good results for Ala, Ile-γ2, Met, and Val methyl groups. Residual protonation in the case of Ile-δ1 and Thr methyl groups leads to artifacts arising from 1H homonuclear scalar coupling, but these effects can be easily eliminated. Leu residues for which the methyl carbon is strongly scalar coupled with the adjacent carbon spin remain problematic, and, even though the use of a special pulse scheme can avoid these artifacts, this only works over a limited bandwidth, making the approach nonoptimal for those methyl groups in proteins. To resolve this issue, we favor Otten et al. an approach where [U-1H,1-13C]-D-glucose is used as carbon source, thereby eliminating strong 13C scalar couplings in Leu residues altogether. The application to the N-terminal receiver domain of the Nitrogen Regulatory Protein C (NtrCr) demonstrates that the experiment can be used to characterize exchange processes in the microsecond time scale, because high effective ﬁeld strengths can be attained by using 1H CPMG pulse trains. The fact that methyl groups, which exhibit narrow lines and high sensitivity, are used as probes to detect dynamics suggests a wide range of applications, including higher molecular weight systems, as well as temperature-dependent studies to characterize the thermo- dynamics of enzyme dynamics. Acknowledgment. We thank Eva Thulin and Prof. Dr. Mikael Akke (University of Lund) for the gift of the pRCB1 expression vector for calbindin D9k and Auke van Heel for preparing the calbindin D9k sample in ∼100% D2O. We are grateful to Drs. Pramodh Vallurupalli, Flemming Hansen, Ranjith Muhandiram, and Lewis Kay for sharing their Varian NMR pulse sequence codes. Dr. Marco Tessari (Radboud University Nijmegen) is gratefully acknowledged for providing measurement time on the 800 MHz spectrometer and his help in setting up the experiments. We thank Dr. Ruud Scheek (University of Groningen) for helpful discussions about data ﬁtting and error analysis. This work was supported by a VIDI grant to F.A.A.M. from The Netherlands Organization for Scientiﬁc Research (NWO), the Howard Hughes Medical Institute (HHMI) to D.K., and a DOE grant (Ofﬁce of Basic Energy Sciences) to D.K. (DE-FG02-05ER15699). Supporting Information Available: A more detailed descrip- tion of experimental scheme C, one ﬁgure showing all 1H CPMG relaxation dispersion proﬁles of calbindin D9k, one ﬁgure showing the 1H CPMG relaxation dispersion proﬁles (at 600 and 800 MHz) for all methyl groups in NtrCr showing exchange, one table with the effective ﬁeld strengths used in the 1H CPMG relaxation dispersion experiments on NtrCr, and one table listing the exchange parameters obtained from ﬁtting the NtrCr data on both ﬁelds on a per-residue basis. This material is available free of charge via the Internet at http://pubs.acs.org. (95) The PyMOL Molecular Graphics System, version 1.3, Schro¨dinger, LCC. JA107410X 17014 J. AM. CHEM. SOC. 9 VOL. 132, NO. 47, 2010