Specific 12CβD212CγD2S13CεHD2 IsotopomerLabeling of Methionine ToCharacterize Protein Dynamics by 1H and 13CNMR Relaxation Dispersion

摘要

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 relaxationdispersion for the nine methionines in calcium-free calmodulin (apo-CaM).The results show that the C-terminal domain, but not the N-terminaldomain, of apo-CaM undergoes fast exchange between the ground stateand a high-energy state. Since target proteins are known to bind specificallyto the C-terminal domain of apo-CaM, we speculate that the high-energystate might be involved in target binding through conformational selection.