Independent movement of the regulatory and catalytic domains of myosin heads revealed by phosphorescence anisotropy

Inter- and intradomain flexibility of the myosin head was measured using ph osphorescence anisotropy of selectively labeled parts of the molecule. Whol e myosin and the myosin head, subfragment-l (S1), were labeled with eosin-5 -iodoacetamide on the catalytic domain (Cys 707) and on two sites on the re gulatory domain (Cys 177 on the essential light chain and Cys 154 on the re gulatory light chain). Phosphorescence anisotropy was measured in soluble S 1 and myosin, with and without F-actin, as well as in synthetic myosin fila ments. The anisotropy of the former were too low to observe differences in the domain mobilities, including when bound to actin, However, this was not the case in the myosin filament. The final anisotropy of the probe on the catalytic domain was 0.051, which increased for probes bound to the essenti al and regulatory light chains to 0.085 and 0.089, respectively. These diff erences can be expressed in terms of a "wobble in a cone" model, suggesting various amplitudes. The catalytic domain was least restricted, with a 51 /- 5 degrees half-cone angle, whereas the essential and regulatory light ch ain amplitude was less than 29 degrees. These data demonstrate the presence of a point of flexibility between the catalytic and regulatory domains. Th e presence of the "hinge" between the catalytic and regulatory domains, wit h a rigid regulatory domain, is consistent with both the "swinging lever ar m" and "'Brownian ratchet" models of force generation. However, in the form er case there is a postulated requirement for the hinge to stiffen to trans mit the generated torque associated by nucleotide hydrolysis and actin bind ing.