Specific 12CβD212CγD2S13CεHD2 Isotopomer Labeling of Methionine To Characterize Protein Dynamics by 1H and 13C NMR Relaxation Dispersion
Open Access
- 2 November 2012
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 134 (45), 18562-18565
- https://doi.org/10.1021/ja309294u
Abstract
Protein dynamics on the micro- to millisecond time scale is increasingly found to be critical for biological function, as demonstrated by numerous NMR relaxation dispersion studies. Methyl groups are excellent probes of protein interactions and dynamics because of their favorable NMR relaxation properties, which lead to sharp signals in the 1H and 13C NMR spectra. Out of the six different methyl-bearing amino acid residue types in proteins, methionine plays a special role because of its extensive side-chain flexibility and the high polarizability of the sulfur atom. Methionine is over-represented in many protein–protein recognition sites, making the methyl group of this residue type an important probe of the relationships among dynamics, interactions, and biological function. Here we present a straightforward method to label methionine residues with specific 13CHD2 methyl isotopomers against a deuterated background. The resulting protein samples yield NMR spectra with improved sensitivity due to the essentially 100% population of the desired 13CHD2 methyl isotopomer, which is ideal for 1H and 13C spin relaxation experiments to investigate protein dynamics in general and conformational exchange in particular. We demonstrate the approach by measuring 1H and 13C CPMG relaxation dispersion for the nine methionines in calcium-free calmodulin (apo-CaM). The results show that the C-terminal domain, but not the N-terminal domain, of apo-CaM undergoes fast exchange between the ground state and a high-energy state. Since target proteins are known to bind specifically to the C-terminal domain of apo-CaM, we speculate that the high-energy state might be involved in target binding through conformational selection.This publication has 59 references indexed in Scilit:
- Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersionJournal of Biomolecular NMR, 2012
- Transient, Sparsely Populated Compact States of Apo and Calcium-Loaded Calmodulin Probed by Paramagnetic Relaxation Enhancement: Interplay of Conformational Selection and Induced FitJournal of the American Chemical Society, 2011
- Structural and Energetic Determinants of Apo Calmodulin Binding to the IQ Motif of the NaV1.2 Voltage-Dependent Sodium ChannelStructure, 2011
- Solution NMR Structure of Apo-Calmodulin in Complex with the IQ Motif of Human Cardiac Sodium Channel NaV1.5Journal of Molecular Biology, 2011
- The dynameomics rotamer library: Amino acid side chain conformations and dynamics from comprehensive molecular dynamics simulations in waterProtein Science, 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 NtrCrJournal of the American Chemical Society, 2010
- Intrinsically Disordered PEP-19 Confers Unique Dynamic Properties to Apo and Calcium CalmodulinBiochemistry, 2010
- Thermodynamic Effects of Noncoded and Coded Methionine Substitutions in CalmodulinBiophysical Journal, 2009
- Measurement of bond vector orientations in invisible excited states of proteinsProceedings of the National Academy of Sciences of the United States of America, 2007
- Characterization of Chemical Exchange Using Residual Dipolar CouplingJournal of the American Chemical Society, 2007