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Lysine methylation strategies for characterizing protein conformations by NMR

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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, protein–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 13C–2H scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance 13C signal.

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Acknowledgments

RSP acknowledges NSERC (Grant number 261980) for a research discovery award. MPB acknowledges support from the Stanford Medical Scientist Training Program.

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Authors and Affiliations

  1. Department of Chemical and Physical Sciences, University of Toronto, UTM, 3359 Mississauga Rd. North, Mississauga, ON, L5L 1C6, Canada

    Sacha Thierry Larda, Ferenc Evanics & R. Scott Prosser

  2. Department of Anesthesia and Perioperative Care, University of California, San Francisco, CA, 94143, USA

    Michael P. Bokoch

  3. Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada

    R. Scott Prosser

Authors
  1. Sacha Thierry Larda
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  2. Michael P. Bokoch
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  3. Ferenc Evanics
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  4. R. Scott Prosser
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Corresponding author

Correspondence to R. Scott Prosser.

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10858_2012_9664_MOESM1_ESM.doc

Table S1 reports 1H and 13C chemical shifts and linewidths for methylated HEWL.. Figures S2-S7 detail the effect of pH on chemical shifts of mono- and di-methyl lysines. Figure S8 shows the effect of aldehyde stoichiometry on the resulting ratio of mono- to dimethyl lysines for reductively methylated HEWL. Figure S9 and S10 show the effect of aldehyde stoichiometry on the ratio of monoto dimethyl lysines for reductively methylated BSA (DOC 6083 kb)

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Larda, S.T., Bokoch, M.P., Evanics, F. et al. Lysine methylation strategies for characterizing protein conformations by NMR. J Biomol NMR 54, 199–209 (2012). https://doi.org/10.1007/s10858-012-9664-z

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  • DOI: https://doi.org/10.1007/s10858-012-9664-z

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