A New Rapid Freezing Apparatus for Electron Microscopy

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The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P^3-1-(2-nitropheny1)ethy1 ester of ATP (caged ATP) was analyzed with a newly-developed freezing system using liquid helium. Actin and S1 (100muM each) formed a rope-like double helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. At 15 ms after photolysis, the rope-like double helix was partially disintegrated. The number of S1 attached to actin filaments gradually decreased up to 35 ms after photolysis, and no more changes were detected from 35 to 200 ms. After depletion of ATP. the rope-like double helix was reformed. Taking recent analyses of actomyosin kinetics into consideration, we concluded that most S1 observed on actin filaments at 25-200 ms are so called "weakly-bound S1" (S1.ATP or S1.ADP.Pi) and that the weakly-bound S1 under a rapid association-dissociation equilibrium with actin filaments can be captured by electron microscopy by means of our newly-developed freezing system.This enabled us to directly compare the conformation of weakly- and strongly-bound S1. Within the resolution of deep-etch replica technique, there were no significant conformational differences between weakly- and strongly-bound S1, and neither types of S1 showed any positive cooperativity in their binding to actin filaments. Close comparison revealed that the weakly- and strongly-bound S1 have different angles of attachment. As compared to strongly-bound S1, weakly-bound S1 showed broad distribution of attachment angle and a decreased tilt from the perpendicular to the filaments. These results discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.