We have used electron paramagnetic probes attached to the
ribose of
ATP (
SL-ATP) to monitor conformational changes in the
nucleotide pocket of
myosin. Spectra for analogs bound to
myosin in the absence of actin showed a high degree of immobilization, indicating a closed
nucleotide pocket. In the Actin.Myosin.SL-
AMPPNP, Actin.Myosin.SL-ADP.BeF(3), and Actin.Myosin.SL-ADP.AlF(4) complexes, which mimic weakly binding states near the beginning of the power
stroke, the
nucleotide pocket remained closed. The spectra of the strongly bound Actin.Myosin.SL-
ADP complex consisted of two components, one similar to the closed pocket and one with increased probe mobility, indicating a more open pocket, The temperature dependence of the spectra showed that the two conformations of the
nucleotide pocket were in equilibrium, with the open conformation more favorable at higher temperatures. These results, which show that opening of the pocket occurs only in the strongly bound states, appear reasonable, as this would tend to keep
ADP bound until the end of the power
stroke. This conclusion also suggests that force is initially generated by a
myosin with a closed
nucleotide pocket.