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• W2135771489 endingPage "17014" @default.
• W2135771489 startingPage "17004" @default.
• W2135771489 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 μs). 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." @default.
• W2135771489 created "2016-06-24" @default.
• W2135771489 creator A5041024225 @default.
• W2135771489 creator A5053909819 @default.
• W2135771489 creator A5058945791 @default.
• W2135771489 creator A5071376755 @default.
• W2135771489 date "2010-11-08" @default.
• W2135771489 modified "2023-10-15" @default.
• W2135771489 title "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 NtrC^<i>r</i>" @default.
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• W2135771489 doi "https://doi.org/10.1021/ja107410x" @default.
• W2135771489 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2991065" @default.
• W2135771489 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21058670" @default.

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