Ca(2+)- and s1-induced movement of troponin T on mutant thin filaments reconstituted with functionally deficient mutant tropomyosin.
The deletion mutant (D234Tm) of rabbit skeletal muscle alpha-tropomyosin, in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing, inhibits the thin filament activated myosin-ATPase activity whether Ca(2+) ion is present or not [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056]. Fluorescence resonance energy transfer (FRET) showed substantial changes in distances between Cys-60 or 250 of troponin T (TnT) and Gln-41 or Cys-374 of actin on wild-type thin filaments corresponding to three states of thin filaments [Kimura et al. (2002) J. Biochem. 132, 93-102]. Troponin T movement on mutant thin filaments reconstituted with D234Tm was compared with that on wild-type thin filaments to understand from which the functional deficiency of mutant thin filaments derives. The Ca(2+)-induced changes in distances between Cys-250 of TnT and Gln-41 or Cys-374 of F-actin were smaller on mutant thin filaments than on wild-type thin filaments. On the other hand, the distances between Cys-60 of TnT and Gln-41 or Cys-374 of F-actin on mutant thin filaments did not change at all regardless of whether Ca(2+) was present. Thus, FRET showed that the Ca(2+)-induced movement of TnT was severely impaired on mutant thin filaments. The rigor binding of myosin subfragment 1 (S1) increased the distances when the thin filaments were fully decorated with S1 in the presence and absence of Ca(2+). However, plots of the extent of S1-incuced movement of TnT against molar ratio of S1 to actin in the presence and absence of Ca(2+) showed that the S1-induced movement of TnT was also impaired on mutant thin filaments. The deficiency of TnT movement on mutant thin filaments causes the altered S1-induced movement of TnI, and mutant thin filaments consequently fail to activate the myosin-ATPase activity even in the presence of Ca(2+).
Kimura
Chieko
C
Department of Applied Chemistry and Biotechnology, Fukui University, Bunkyo, Fukui 910-8507, Japan.
Maeda
Kayo
K
Hai
Hong
H
Miki
Masao
M
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D009282:Naphthalenesulfonates / Q000378:metabolism; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism*; D020107:Troponin T / Q000378:metabolism*
D000199:Actins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D014335:Tropomyosin; D020107:Troponin T; C048876:1,5-AEDANS; D003545:Cysteine; D002118:Calcium