Metal ion and drug binding to proteolytic fragments of calmodulin: proteolytic, cadmium-113, and proton nuclear magnetic resonance studies.
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 three peptides each of the amino- and carboxy-terminal halves of CaM of slightly different length. A purification scheme for the three carboxy-terminal half-peptides is reported. Proton nuclear magnetic resonance (1H NMR) studies of peptides comprising the amino- or carboxy-terminal half of CaM reveal the great structural similarity between these two proteolytic fragments and the intact protein. Since this was observed for the apoprotein as well as the Ca2+-saturated protein, this means that the two 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 two high-affinity binding sites. Furthermore, addition of Ca2+ or Cd2+ induces qualitatively similar changes in the spectra, thus 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 of calmodulin revealed the presence of two distinct drug binding sites, one located in the amino-terminal half and one located 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.
D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D013737:Testis; D013917:Thrombin; D014268:Trifluoperazine / Q000378:metabolism*; D014357:Trypsin
D002147:Calmodulin; D010446:Peptide Fragments; D002104:Cadmium; D014268:Trifluoperazine; D014357:Trypsin; D013917:Thrombin; D002118:Calcium