The rates of switching movement of troponin T between three states of skeletal muscle thin filaments determined by fluorescence resonance energy transfer.
Troponin (Tn) plays the key roles in the regulation of striated muscle contraction. Tn consists of three subunits (TnT, TnC, and TnI). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to Cys-60 or Cys-250 of TnT and Cys-374 of actin was measured to determine the rates of switching movement of the troponin tail domain (Cys-60) and of the TnT-TnI coiled-coil C terminus (Cys-250) between three states (relaxed, closed, and open) of the thin filament. When the free Ca(2+) concentration was rapidly changed, these domains moved with rates of approximately 450 and approximately 85 s(-1) at pH 7.0 on Ca(2+) up and down, respectively. When myosin subfragment 1 (S1) was dissociated from thin filaments by rapid mixing with ATP, these domains moved with a single rate constant of approximately 400 s(-1) in the presence and absence of Ca(2+). The light scattering measurements showed that ATP-induced S1 dissociation occurred with a rate constant >800 s(-1). When S1 was rapidly mixed with the thin filament, these domains moved with almost the same or slightly faster rates than those of S1 binding measured by light scattering. In most but not all aspects, the rates of movement of the troponin tail domain and of the TnT-TnI coiled-coil C terminus were very similar to those of certain TnI sites (N terminus, Cys-133, and C terminus) previously characterized (Shitaka, Y., Kimura, C., Iio, T., and Miki, M. (2004) Biochemistry 43, 10739-10747), suggesting that a series of conformational changes in the Tn complex during switching on or off process occurs synchronously.
Shitaka
Yuji
Y
Department of Applied Chemistry and Biotechnology, Fukui University, 3-9-1 Bunkyo, Fukui 910-8507, Japan.
Kimura
Chieko
C
Miki
Masao
M
D000199:Actins / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D018482:Muscle, Skeletal / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012542:Scattering, Radiation; D013053:Spectrophotometry; D013997:Time Factors; D020107:Troponin T / Q000378:metabolism*
D000199:Actins; D020107:Troponin T; D000255:Adenosine Triphosphate; D003545:Cysteine; D002118:Calcium