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A study has been made of the intrinsic fluorescence associated with G-and F-actin, with EDTA-and heat-denatured actin, and with actin in 8 M urea. A small decrease in the Trp and an increase in the Tyr contribution is associated with the polymerization of G-to F-actin. An appreciable red shift of the fluorescence spectrum occurs when G-or F-actin is denatured, indicating increased exposure of Trp to the aqueous environment, This change in fluorescence produced by the addition of EDTA can be used as a quick measure of the fraction of denatured species associated with a given actin preparation. Attempts to regenerate native actin from denatured actin have been unsuccessful. The fluorescence A ctin, one of the major protein components of the myofibril involved in the process of muscle contraction, is capable of existing in different states. G-actin, the monomeric native form of actin, has a tightly bound nucleotide and a tightly bound Ca2+ or Mgz+ normally found associated with it. If the nucleotide or metal ion is removed, the protein irreversibly denatures (Asakura, 1961 ;Maruyama and Gergely, 1961). This denatured form of actin (d-actin) appears, on the basis of optical rotatory dispersion and circular dichroism studies (Nagy and Jencks, 1962;, to be only partially unfolded compared with actin in 8 M urea (u-actin). F-actin, the fibrous form which exists in the muscle, can be formed from G-actin but not form d-actin by a reversible salt-induced polymerization. Although this reversible polymerization does not appear to have a major role in muscle contraction, localized dislocations of the actin filament may have some importance (Oosawa et al., 1961 ;Asakura et al., 1963;Szent-Gyorgyi and Prior, 1966).The purpose of the present study is to characterize the intrinsic fluorescence properties of the different states of actin in order to further obtain information regarding conformational differences. We found that small differences exist between G-and F-actin with respect to the contributions of both Tyr and Trp to the intrinsic fluorescence when proper corrections for light scattering were made. Studies of the temperature dependence of the fluorescence indicate qualitative and quantitative differences between G-and F-actin in their stability toward heat denaturation. The large fluorescence spectral shift that occurs when G-or F-actin is denatured indicates an increase in average exposure of Trp side chains to the aqueous solvent. The quenching of the fluorescence of G-and F-actin due to the binding of Cu2+ appears to involve energy transfer to a new absorption band associated with the

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].

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