Replacement of Lys-75 of calmodulin affects its interaction with smooth muscle caldesmon.
The interaction of smooth muscle caldesmon with synthetic calmodulin (SynCam) and its five mutants with replacement of Lys-75 was analyzed by means of intrinsic Trp fluorescence, zero-length crosslinking and by caldesmon-induced inhibition of actomyosin ATPase activity. SynCam and its double mutant with replacement K75P and simultaneous insertion of KGK between residues 80 and 81 have a comparably low affinity to caldesmon and the probability of crosslinking of this mutant to caldesmon was the lowest among all mutants analyzed. SynCam and its double mutant (K75P+KGK) induced nearly complete reversion of caldesmon inhibition of actomyosin ATPase activity with half-maximal reversion achieved at about 1 microM. Two mutants, K75A and K75V, with partially stabilized less positive central domain have higher affinity to caldesmon. These mutants induce 80-85% reversion of caldesmon inhibition of actomyosin ATPase and the half-maximal reversion was achieved at about 0.3-0.4 microM. Two last mutants, K75P and K75E, with distorted central domain have high affinity to caldesmon and the probability of crosslinking of K75P to caldesmon was the highest among calmodulin mutants tested. These mutants induced complete reversion of caldesmon inhibition with half-maximal effect observed at 0.3-0.4 microM. We suggest that the length, flexibility and charge of the central domain affect binding of calmodulin mutants and their ability to reverse caldesmon-induced inhibition of actomyosin ATPase activity.
Medvedeva
M V
MV
Department of Biochemistry, School of Biology, Mascow State University, Russia.
Djemuchadze
D R
DR
Watterson
D M
DM
Marston
S B
SB
Gusev
N B
NB
D019943:Amino Acid Substitution; D000818:Animals; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D004372:Ducks; D008239:Lysine / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000037:antagonists & inhibitors; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D009218:Myosins; D008239:Lysine