title: Mutation of Lys-75 affects calmodulin conformation.
abstract: Some properties of synthetic calmodulin and its five mutants with replacement of Lys-75 were analyzed by means of electrophoresis, limited proteolysis and MALDI mass-spectrometry. A double mutant of calmodulin containing insert KGK between residues 80 and 81 and replacement of Lys-75 by Pro has a highly flexible central helix which is susceptible to trypsinolysis in the presence of Ca2+. Two mutants, K75P and K75E, having a distorted central helix demonstrate high resistance to trypsinolysis in the absence of Ca2+. Arg-90 and Arg-106 being the primary site of trypsinolysis of synthetic calmodulin are partially-protected in K75P and K75E mutants. The central helix of K75A and K75V mutants is stabilized by hydrophobic interactions between residues located in positions 71, 72 and 75. In the presence of Ca2+, the central helix of K75V is resistant to trypsinolysis. Mutations K75A and K75V decrease the rate of trypsinolysis of the central helix with a simultaneous increase of the rate of trypsinolysis in the C-terminal domain of calmodulin. It is concluded that the point mutation in the central helix has a long distance effect on the structure of calmodulin.
authors.affiliation: Department of Biochemistry, School of Biology, Moscow State University, Russia.
authors.forename: M V
authors.initials: MV
authors.lastname: Medvedeva
authors.forename: O V
authors.initials: OV
authors.lastname: Polyakova
authors.forename: D M
authors.initials: DM
authors.lastname: Watterson
authors.forename: N B
authors.initials: NB
authors.lastname: Gusev
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D014357:Trypsin / Q000737:chemistry
chemical_list: D002147:Calmodulin; D010446:Peptide Fragments; D014357:Trypsin; D008239:Lysine; D002118:Calcium