Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: Biochemical and spectroscopic studies

Calmodulin (CaM) is a 148-residue regulatory calcium-binding protein that a ctivates a wide range of target proteins and enzymes. Calcium-saturated CaM has a bilobal structure, and each domain has an exposed hydrophobic surfac e region where target proteins are bound. These two "active sites" of calmo dulin are remarkably rich in Met residues. Here we have biosynthetically su bstituted (up to 90% incorporation) the unnatural amino acids ethionine (Et h) and norleucine (Nle) for the nine Met residues of CaM. The substituted p roteins bind in a calcium-dependent manner to hydrophobic matrices and a sy nthetic peptide, encompassing the CaM-binding domain of myosin light-chain kinase (MLCK). Infrared and circular dichroism spectroscopy show that there are essentially no changes in the secondary structure of these proteins co mpared to wild-type CaM (WT-CaM). One- and two-dimensional NMR studies of t he Eth-CaM and Nle-CaM proteins reveal that, while the core of the proteins is relatively unaffected by the substitutions, the two hydrophobic interac tion surfaces adjust to accommodate the Eth and Nle residues. Enzyme activa tion studies with MLCK show that Eth-CaM and Nle-CaM activate the enzyme to 90% of its maximal activity, with little changes in dissociation constant. For calcineurin only 50% activation was obtained, and the K-D for Nle-CaM also increased 3.5-fold compared with WT-CaM. These data show that the "act ive site" Met residues of CaM play a distinct role in the activation of dif ferent target enzymes, in agreement with site-directed mutagenesis studies of the Met residues of CaM.