CONFORMATION OF MYOSIN - EFFECTS OF SUBSTRATE AND MODIFIERS

Myosin was studied with a macromolecular probe, 8-anilino-1-naphthalene-sulfonate (ANS). The fluorescence of this probe bound to myosin when the protein-ligand complex was excited at 280 nm suffered a small reversible decrease in the presence of ATP (Ca2-, 0.6 M KCl, pH 8) which was correlated with the kinetics of hydrolysis. The time required for the fluorescence to return was proportional to the enzyme concentration at a fixed ATP concentration. Measurements of myosin ATPase under similar conditions showed that the restoration of the fluorescence to its original level began when the ATPase activity began to decrease from its steady-state level, and ended when ATP was depleted. This transition is attributed to a change in localized structure of myosin, as reflected by a change in the transfer of excitation energy from myosin to the bound probe. The effects of urea, alkaline pH, N-ethylmaleimide, and iodoacetamide on the fluorescence properties of bound ANS were also studied. The results are consistent with the view that the loss of myosin-ATPase activity caused by these agents is associated with an extensive change of the myosin conformation. The fluorescence enhancement of the probe bound to native myosin, and to myosin modified with p-chloromercuribenzoate was investigated in 3 M urea as a function of time. The enhancement increased as a first-order process. The rate constants increase in the order: native myosin ATP, native myosin, and modified myosin. These results suggest that in urea solution the native enzyme may be thermodynamically more stable than the modified enzyme, and that ATP stabilizes myosin with respect to denaturation by urea. The difference in stability can be attributed to a difference in conformation. © 1969.