Lysine methylation strategies for characterizing protein conformations by NMR

In the presence of formaldehyde and a mild reducing agent, reductive methylation is known to achieve near complete dimethylation of protein amino groups under non-denaturing conditions, in aqueous media. Amino methylation of proteins is employed in mass spectrometric, crystallographic, and NMR studies. Where biosynthetic labeling is prohibitive, amino C-13-methylation provides an attractive option for monitoring folding, kinetics, protein-protein and protein-DNA interactions by NMR. Here, we demonstrate two improvements over traditional C-13-reductive methylation schemes: (1) By judicious choice of stoichiometry and pH, epsilon-aminos can be preferentially monomethylated. Monomethyl tags are less perturbing and generally exhibit improved resolution over dimethyllysines, and (2) By use of deuterated reducing agents and C-13-formaldehyde, amino groups can be labeled with (CH2D)-C-13 tags. Use of deutero-C-13-formaldehyde affords either (CHD2)-C-13, or (CD3)-C-13 probes depending on choice of reducing agent. Making use of C-13-H-2 scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance C-13 signal.