Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins.
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.
Abraham
Sherwin J
SJ
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.
Kobayashi
Tomoyoshi
T
Solaro
R John
RJ
Gaponenko
Vadim
V
D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008239:Lysine / Q000737:chemistry*; D008745:Methylation; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism
D002147:Calmodulin; D002247:Carbon Isotopes; D011506:Proteins; D019209:Troponin C; D005982:Glutathione Transferase; D009113:Muramidase; D008239:Lysine; D002118:Calcium