Mutation of Lys-75 affects calmodulin conformation

Some properties of synthetic calmodulin and its five mutants with replaceme nt of Lys-75 mere analyzed by means of electrophoresis, limited proteolysis and MALDI mass-spectrometry, A double mutant of calmodulin containing inse rt KGK between residues 80 and 81 and replacement of Lys-75 by Pro has a hi ghly flexible central helix which is susceptible to trypsinolysis in the pr esence of Ca2+. Two mutants, K75P and K75E, having a distorted central heli x demonstrate high resistance to trypsinolysis in the absence of Ca2+. Arg- 90 and Arg-106 being the primary site of trypsinolysis of synthetic calmodu lin are partially-protected in K75P and K75E mutants. The central helix of K75A and K75V mutants is stabilized by hydrophobic interactions between res idues located in positions 71, 72 and 75, In the presence of Ca2+, the cent ral helix of K75V is resistant to trypsinolysis, Mutations K75A and K75V de crease the rate of trypsinolysis of the central helix with a simultaneous i ncrease 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 d istance effect on the structure of calmodulin, (C) 1999 Federation of Europ ean Biochemical Societies.