Cooperativity and switching within the three-state model of muscle regulation.
Thin filament regulation is mediated by the presence of tropomyosin (Tm) and troponin (Tn) on the actin filament. Binding of Tm alone induces two states, closed and open (with the equilibrium between them defined by KT), which differ in their affinity for myosin subfragment 1 (S1). Cooperative switching between the states results in characteristic sigmoidal myosin S1 binding curves. In the presence of Tn and absence of Ca2+, a third state, blocked, has previously been kinetically shown to be present, leading to the three state model of McKillop and Geeves [(1993) Biophys. J. 65, 693-701]. We have measured equilibrium binding of S1 to phalloidin-stabilized pyrene-actin filaments by monitoring the pyrene fluorescence at 50 nM, a concentration 10-fold lower than previously possible. In combination with kinetic studies, we show that the data can be fitted to a modified version of the three-state model with an additional term allowing for a varying apparent cooperative unit size (n). Our results show that the apparent cooperative unit size (n) is dependent upon both the presence of Tn and of Ca2+. Also in the absence of Ca2+, the occupancy of the blocked state (defined by KB) is accompanied by a 2-3-fold reduction in KT. These results are discussed in comparison to the Hill model [(1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190] and a flexible model of thin filament regulation based upon that of Lehrer et al. [(1997) Biochemistry 36, 13449-13455].
Maytum
R
R
Max Planck Institut für Moleculare Physiologie, Dortmund, Germany.
Lehrer
S S
SS
Geeves
M A
MA
D000199:Actins / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000737:chemistry*; D015879:Myosin Subfragments / Q000737:chemistry; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000737:chemistry; D014336:Troponin / Q000737:chemistry
D000199:Actins; D015879:Myosin Subfragments; D014335:Tropomyosin; D014336:Troponin; D000244:Adenosine Diphosphate; D002118:Calcium