Lysine methylation strategies for characterizing protein conformations by NMR

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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 13C-methylation provides an attractive option for monitoring folding, kinetics, proteinâ??protein and protein-DNA interactions by NMR. Here, we demonstrate two improvements over traditional 13C-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 13C-formaldehyde, amino groups can be labeled with 13CH2D tags. Use of deutero-13C-formaldehyde affords either 13CHD2, or 13CD3 probes depending on choice of reducing agent. Making use of 13Câ??2H scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance 13C signal.

• Content Type Journal Article
• Category Article
• Pages 1-11
• DOI 10.1007/s10858-012-9664-z
• Authors
  • Sacha Thierry Larda, Department of Chemical and Physical Sciences, University of Toronto, UTM, 3359 Mississauga Rd. North, Mississauga, ON L5L 1C6, Canada
  • Michael P. Bokoch, Department of Anesthesia and Perioperative Care, University of California, San Francisco, CA 94143, USA
  • Ferenc Evanics, Department of Chemical and Physical Sciences, University of Toronto, UTM, 3359 Mississauga Rd. North, Mississauga, ON L5L 1C6, Canada
  • R. Scott Prosser, Department of Chemical and Physical Sciences, University of Toronto, UTM, 3359 Mississauga Rd. North, Mississauga, ON L5L 1C6, Canada

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR