Abstract
Reductive methylation of lysine residues in proteins offers a way to introduce 13C methyl groups into otherwise unlabeled molecules. The 13C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of 13C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca2+-bound Calmodulin, Lysozyme, Ca2+-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of 13C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment.
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Abbreviations
- CaM:
-
Calmodulin
- HSQC:
-
Heteronuclear Single Quantum Coherence
- GST:
-
Glutathione S-Transferase
- TFP:
-
Trifluoperazine
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Acknowledgements
We are grateful to Dr. Arnon Lavie and Ms. Ming F. Lye for providing purified GST for our experiments. The research was supported by the American Cancer Society, Illinois Division Grant ACS 08-14 to Vadim Gaponenko, NIH Grant R01HL082923 to Tomoyoshi Kobayashi, and P01HL62426 to R. John Solaro. NMR spectra were recorded using the NMR facility at the University of Illinois at Chicago funded by the NIH Grant P41 GM68944 to Peter GW Gettins.
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Abraham, S.J., Kobayashi, T., John Solaro, R. et al. Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins. J Biomol NMR 43, 239–246 (2009). https://doi.org/10.1007/s10858-009-9306-2
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DOI: https://doi.org/10.1007/s10858-009-9306-2