title: Lysine methylation strategies for characterizing protein conformations by NMR.
abstract: 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 (13)C-methylation provides an attractive option for monitoring folding, kinetics, protein-protein and protein-DNA interactions by NMR. Here, we demonstrate two improvements over traditional (13)C-reductive 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 (13)C-formaldehyde, amino groups can be labeled with (13)CH(2)D tags. Use of deutero-(13)C-formaldehyde affords either (13)CHD(2), or (13)CD(3) probes depending on choice of reducing agent. Making use of (13)C-(2)H scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance (13)C signal.
authors.affiliation: Department of Chemical and Physical Sciences, University of Toronto, UTM, 3359 Mississauga Rd. North, Mississauga, ON L5L 1C6, Canada. s.larda@utoronto.ca
authors.forename: Sacha Thierry
authors.initials: ST
authors.lastname: Larda
authors.forename: Michael P
authors.initials: MP
authors.lastname: Bokoch
authors.forename: Ferenc
authors.initials: F
authors.lastname: Evanics
authors.forename: R Scott
authors.initials: RS
authors.lastname: Prosser
mesh_terms: D002247:Carbon Isotopes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008745:Methylation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D002247:Carbon Isotopes; D011506:Proteins; D008239:Lysine