Determination of the side chain pKa values of the lysine residues in calmodulin.
The 7 Lys residues in mammalian calmodulin (CaM) were reductively methylated with 13C-enriched formaldehyde and studied by (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR. The apo- and Ca(2+)-forms of CaM, as well as a complex with a 22-residue peptide which comprises the CaM binding region of myosin light chain kinase were studied. The complete assignment of the two-dimensional NMR spectra was obtained by site-directed mutagenesis (Lys-->Gln) of all the Lys. The pKa values for the individual Lys could be determined by pH titration experiments. In Ca(2+)-CaM, the pKa values range from 9.29 (Lys-75) to 10.23 (Lys-77). The Lys in apo-CaM have higher pKa values than those in Ca(2+)-CaM. The binding of the myosin light chain kinase peptide gives rise to an increase of the pKa values of Lys-148 and Lys-75 by 0.5 and 0.8 pH units, respectively; this results from the relocation of their side chains to a completely solvent accessible state. The changes in the pKa values upon binding Ca2+ or the myosin light chain kinase peptide show a remarkable correlation with earlier reported chemical reactivity changes. Thus, our results indicate that pKa values, rather than structural and steric effects, play the dominant role in determining the reactivity of Lys side chains towards small electrophilic chemical modification reagents. The methodology used here could prove useful for the determination of individual pKa values in other proteins.
Zhang
M
M
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Vogel
H J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013737:Testis / Q000378:metabolism
D002147:Calmodulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009219:Myosin-Light-Chain Kinase; D008239:Lysine; D002118:Calcium