Cooperativity and switching within the three-state model of muscle regulation

Thin filament regulation is mediated by the presence of tropomyosin (Tm) an d troponin (Tn) on the actin filament. Binding of Tm alone induces two stat es, closed and open (with the equilibrium between them defined by K-T), whi ch differ in their affinity for myosin subfragment 1 (S1). Cooperative swit ching between the states results in characteristic sigmoidal myosin S1 bind ing curves. In the presence of Tn and absence of Ca2+, a third state, block ed, has previously been kinetically shown to be present, leading to the thr ee state model of McKillop and Geeves [(1993) Biophys. J. 65, 693-701]. We have measured equilibrium binding of S1 to phalloidin-stabilized pyrene-act in filaments by monitoring the pyrene fluorescence at 50 nM, a concentratio n 10-fold lower than previously possible. In combination with kinetic studi es, 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 coopera tive unit size (n). Our results show that the apparent cooperative unit siz e (n) is dependent upon both the presence of Tn and of Ca2+. Also in the ab sence of Ca2+, the occupancy of the blocked state (defined by K-B) is accom panied by a 2-3-fold reduction in K-T. These results are discussed in compa rison 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 Lehre r et al. [(1997) Biochemistry 36, 13449-13455].