Metal ion and drug binding to proteolytic fragments of calmodulin: proteolytic cadmium-113 and proton nuclear magnetic resonance studies

Publisher Website
Google Scholar
Abstract
Tryptic fragmentation of Ca2+-saturated calmodulin (CaM) takes place mainly at Lys-77; however, proteolysis can occur instead at Arg-74 or Lys-75. This cleavage pattern results in the production of 3 peptides each of the amino- and carboxy-terminal halves of CaM of slightly different length. A purification scheme for the 8 carboxy-terminal half-peptides is reported. 1H NMR studies of peptides comprising the amino- or caboxy-terminal half of CaM reveal the great structural similarity between these 2 proteolytic fragments and the intact protein. Since this was observed for the apoprotein as well as the Ca2+-sautrated protein, this means that the 2 halves of the protein are independently folded. A comparison of the changes in the 1H NMR spectra observed for the intact protein and the fragments upon addition of Ca2+ clearly identified sites III and IV as the 2 high-affinty binding sites. Addition of Ca2+ or Cd2+ induces qualitatively similar changes in the spectra, indicating that Cd2+ is a reliable replacement for Ca2+ in these studies. Subsequent 113Cd NMR studies of trifluoperazine (TFP) binding to tryptic and thrombic fragments od calmodulin revealed the presence of 2 distinct drug binding sites, one located in the amino-terminal half and one in the carboxy-terminal half. The spectral changes, induced upon addition of the antipsychotic drug, were similar to those observed upon binding of TFP to intact calmodulin. The strongest TFP binding site is located in the carboxy-terminal half.