Published on Web 11/08/2010 Probing Microsecond Time Scale Dynamics in Proteins by Methyl 1H Carr-Purcell-Meiboom-Gill Relaxation Dispersion NMR Measurements. Application to Activation of the Signaling Protein NtrCr Renee Otten,† Janice Villali,‡ Dorothee Kern,‡ and Frans A. A. Mulder*,† Groningen Biomolecular Sciences and Biotechnology Institute, UniVersity of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands, and Department of Biochemistry and Howard Hughes Medical Institute, Brandeis UniVersity, Waltham, Massachusetts 02452, United States Received August 17, 2010; E-mail: f.a.a.mulder@rug.nl Abstract: To study microsecond processes by relaxation dispersion NMR spectroscopy, low power deposition and short pulses are crucial and encourage the development of experiments that employ 1H Carr-Purcell-Meiboom-Gill (CPMG) pulse trains. Herein, a method is described for the comprehensive study of microsecond to millisecond time scale dynamics of methyl groups in proteins, exploiting their high abundance and favorable relaxation properties. In our approach, protein samples are produced using [1H, 13C]-D-glucose in ∼100% D2O, which yields CHD2 methyl groups for alanine, valine, threonine, isoleucine, leucine, and methionine residues with high abundance, in an otherwise largely deuterated background. Methyl groups in such samples can be sequence-speciﬁcally assigned to near completion, using 13C TOCSY NMR spectroscopy, as was recently demonstrated (Otten, R.; et al. J. Am. Chem. Soc. 2010, 132, 2952-2960). In this Article, NMR pulse schemes are presented to measure 1H CPMG relaxation dispersion proﬁles for CHD2 methyl groups, in a vein similar to that of backbone relaxation experiments. Because of the high deuteration level of methyl-bearing side chains, artifacts arising from proton scalar coupling during the CPMG pulse train are negligible, with the exception of Ile-δ1 and Thr-γ2 methyl groups, and a pulse scheme is described to remove the artifacts for those residues. Strong 13C scalar coupling effects, observed for several leucine residues, are removed by alternative biochemical and NMR approaches. The methodology is applied to the transcriptional activator NtrCr, for which an inactive/active state transition was previously measured and the motions in the microsecond time range were estimated through a combination of backbone 15N CPMG dispersion NMR spectroscopy and a collection of experiments to determine the exchange-free component to the transverse relaxation rate. Exchange contributions to the 1H line width were detected for 21 methyl groups, and these probes were found to collectively report on a local structural rearrangement around the phosphorylation site, with a rate constant of (15.5 ( 0.5) × 103 per second (i.e., τex ) 64.7 ( 1.9 µ s). The affected methyl groups indicate that, already before phosphorylation, a substantial, transient rearrangement takes place between helices 3 and 4 and strands 4 and 5. This conformational equilibrium allows the protein to gain access to the active, signaling state in the absence of covalent modiﬁcation through a shift in a pre-existing dynamic equilibrium. Moreover, the conformational switching maps exactly to the regions that differ between the solution NMR structures of the fully inactive and active states. These results demonstrate that a cost-effective and quantitative study of protein methyl group dynamics by 1H CPMG relaxation dispersion NMR spectroscopy is possible and can be applied to study functional motions on the microsecond time scale that cannot be accessed by backbone 15N relaxation dispersion NMR. The use of methyl groups as dynamics probes extends such applications also to larger proteins. Introduction To understand the function of a biological macromolecule, it is of critical importance to complement a high-resolution three-dimensional structure with a detailed insight in its molecular dynamics.1,2 For a growing number of systems, it has been found that slow (microsecond to millisecond), collective motions can be rate limiting for biological function. † University of Groningen. ‡ Brandeis University. (1) Kay, L. E. Nat. Struct. Biol. 1998, 5, 513–517. (2) Ishima, R.; Torchia, D. A. Nat. Struct. Biol. 2000, 7, 740–743. 17004 9 J. AM. CHEM. SOC. 2010, 132, 17004–17014 Nuclear magnetic resonance (NMR) spectroscopy is an ideal technique for studying dynamical processes on this time scale, being capable of providing such information for many different nuclei with atomic resolution.3-5 Speciﬁcally, microsecond-millisecond dynamics can be studied through NMR relaxation dispersion experiments, and applications to (3) Palmer, A. G.; Massi, F. Chem. ReV. 2006, 106, 1700–1719. (4) Mittermaier, A.; Kay, L. E. Science 2006, 312, 224–228. (5) Mittermaier, A. K.; Kay, L. E. Trends Biochem. Sci. 2009, 34, 601– 611. 10.1021/ja107410x  2010 American Chemical Society