Calcium-induced sensitization of the central helix of calmodulin to proteolysis.
The rate of proteolysis of trypsin-sensitive bonds was used to examine the nature of the structural changes accompanying Ca2+ and Mg2+ binding to calmodulin. In the Ca(2+)-free form, the rates of proteolysis at Arg-106 and Arg-37 are rapid (greater than 300 and 28 nmol min-1 mL-1, respectively), the bonds at Arg-74, Lys-75, and Lys-77, in the central helix, are cleaved more slowly (10 nmol min-1 mL-1), and a lag in the cleavage at the remaining bonds (Lys-13, Lys-30, Arg-86, Arg-90, and Arg-126) suggests that they are not cleaved in the native protein. High concentrations of Ca2+, but not Mg2+, almost completely abolish proteolysis at Arg-106 and drastically reduce the rate of cleavage at Arg-37. Both Ca2+ and Mg2+ exert a moderate protective effect on the proteolysis of the central helix. These results suggest that the F-helix of domains III and, to a lesser extent, the F-helix of domain I are somewhat flexible in the Ca(2+)-free form and are stabilized by Ca2+. Whereas full occupancy of the four Ca(2+)-binding sites produces little change in the susceptibility of the central helix to proteolytic attack, binding of two Ca2+ produces a 10-fold enhancement of the rate of proteolysis in this part of the molecule. We propose that at intermediate Ca2+ levels the flexibility of the central helix of calmodulin is greatly increased, resulting in the transient formation of intermediates which have not been detected by spectroscopic techniques but are trapped by the irreversible action of trypsin.
Mackall
J
J
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Klee
C B
CB
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D014357:Trypsin
D002147:Calmodulin; D014357:Trypsin; D008274:Magnesium; D002118:Calcium