Cooperative binding of myosin subfragment one to regulated actin as measured by fluorescence changes of troponin I modified with different fluorophores.

Binding studies of myosin subfragment one (S-1) to regulated actin in the presence and absence of Ca2+ indicate that, as S-1 binds to regulated actin, tropomyosin-actin units undergo a cooperative transition from a weak to a strong S-1-binding form. Trybus and Taylor (Trybus, K.M., and Taylor, E.W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7209-7213) suggested that this transition could be measured by the change in fluorescence of troponin I modified with 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3-diazole (IANBD). In the present study, this was tested by determining whether the change in fluorescence was proportional to the fraction of tropomyosin-actin units in the strong S-1-binding form as predicted by our model on the cooperative binding of S-1 to regulated actin (Hill, T.L., Eisenberg, E., and Greene, L.E. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190). Experiments were performed both in the presence and absence of Ca2+ by using troponin I modified with either IANBD or 5'-iodoacetamidofluorescein. In the presence of Ca2+, it was found, in agreement with the suggestion of Trybus and Taylor, that the change in fluorescence induced by S-1 was proportional to the fraction of tropomyosin-actin units shifting into the strong S-1 binding form, rather than to the fraction of actin sites having bound S-1. In the absence of Ca2+, the change in fluorescence induced by S-1 also did not reflect the binding of S-1 to regulated actin. However, in contrast to the results in the presence of Ca2+, the change in fluorescence induced by S-1 binding in the absence of Ca2+ was not in agreement with the fraction of tropomyosin-actin units calculated to be in the strong S-1 binding form by the model of Hill et al. Although a more complex model than that of Hill et al. may account for the observed fluorescence changes, it seems equally likely that at least in the absence of Ca2+, the change in fluorescence may be reflecting a more complex behavior than only the transition of tropomyosin-actin units between the weak and strong S-1-binding forms.