Lysine methylation strategies for characterizing protein conformations by NMR

摘要

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 13C-methylation provides an attractive option for monitoring folding, kinetics, protein- protein and protein-DNA interactions by NMR. Here, we demonstrate two improvements over traditional 13Creductive methylation schemes: (1) By judicious choice of stoichiometry and pH, ε-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 13C-formaldehyde, amino groups can be labeled with 13CH 2D tags. Use of deutero- 13C-formaldehyde affords either 13CHD 2, or 13CD 3 probes depending on choice of reducing agent. Making use of 13C- 2H scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance 13C signal.