CHARACTERIZATION OF TRIMETHYLLYSINE-115 IN CALMODULIN BY N-14 AND C-13 NMR-SPECTROSCOPY

In this paper we describe three approaches to study the single trimeth yllysine 115 in calmodulin. First, N-14 NMR spectroscopy has been used as a novel spectroscopic tool. Because of the unique symmetrical tetr ahedral substitution of its side chain, the trimethyllysine residue gi ves rise to a sharp N-14 NMR resonance; hence, this has allowed the de tection and quantitation of the level of trimethylation. Trimethyllysi ne side chains of bovine testis calmodulin and yeast cytochrome c were shown to have a high mobility in aqueous solution as determined by N- 14 NMR relaxation measurements. Second, we have purified mammalian cal modulin from an overproducing Escherichia coli strain. By comparison o f the H-1-C-13 heteronuclear multiple quantum coherence spectra of C-1 3-dimethylated calmodulin samples from bovine testis and E. coli, the resonance for Lys-115 in bacterially expressed calmodulin could be ide ntified. pH titration experiments showed that the epsilon-NH2 group of Lys-115 has a normal pK alpha value both in the apo and Ca2+ forms of the protein and in a complex of calmodulin with a 22-residue calmodul in-binding peptide derived from myosin light chain kinase. Third, we h ave shown that mutation of Lys-115 to the uncharged Gln residue does n ot alter the ability of the protein to stimulate the enzymes cyclic nu cleotide phosphodiesterase and myosin light chain kinase. These result s show that the trimethylation of Lys-115 is not caused by an unusual pK alpha and reactivity of its epsilon-NH2 group and that its side cha in remains flexible. Moreover, our data suggest that the introduction of a permanent positive charge on Lys-115 by trimethylation is also no t the major reason for this specific post-translational modification.