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 activates a wide range of target proteins and enzymes. Calcium-saturated CaM has a bilobal structure, and each domain has an exposed hydrophobic surface region where target proteins are bound. These two "active sites" of calmodulin are remarkably rich in Met residues. Here we have biosynthetically substituted (up to 90% incorporation) the unnatural amino acids ethionine (Eth) and norleucine (Nle) for the nine Met residues of CaM. The substituted proteins bind in a calcium-dependent manner to hydrophobic matrices and a synthetic 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 compared to wild-type CaM (WT-CaM). One- and two-dimensional NMR studies of the Eth-CaM and Nle-CaM proteins reveal that, while the core of the proteins is relatively unaffected by the substitutions, the two hydrophobic interaction surfaces adjust to accommodate the Eth and Nle residues. Enzyme activation 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 "active site" Met residues of CaM play a distinct role in the activation of different target enzymes, in agreement with site-directed mutagenesis studies of the Met residues of CaM.
Yuan
T
T
Department of Biological Sciences, The University of Calgary, Alberta, Canada.
Vogel
H J
HJ
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005001:Ethionine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009646:Norleucine / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared
D000596:Amino Acids; D002147:Calmodulin; D009646:Norleucine; D008715:Methionine; D009219:Myosin-Light-Chain Kinase; D019703:Calcineurin; D005001:Ethionine