A. Orlova and E.H. Egelman
Univ. of Minnesota Medical School, Minneapolis, MN 55455.
Many aspects of cooperative behavior within pure F-actin filaments have been described, by us and by other laboratories. We have shown that with Ca2+ bound at the high-affinity metal binding site in actin, there is a very large cooperativity in the binding of HMM, but no cooperativity for S1. With Mg2+ bound at the high affinity site, or with conditions that stabilize the conformation of subdomain-2 of actin, there is no cooperativity seen with either HMM or S1. These results show that the two heads of HMM can induce structural changes in F-actin that are not observed with the single head of S1. They also support the notion that the binding of myosin to F-actin induces a conformational change in subdomain-2 of actin, and that under certain conditions this conformational change can be cooperatively propagated through an actin filament. An interesting question is whether the structural state of F-actin relaxes after myosin binds and is released, and whether this relaxation time is on a short or long time scale. If the relaxation time is very long (e.g., minutes) it is possible that most of the helical disorder that exists within F-actin may be relatively static. This could reconcile micromechanical measurements of actin's torsional flexibility (Tsuda et al., PNAS 93, 12937-42, 1996) with observations from EM that suggest a larger torsional freedom. To test this, we are using electron microscopy of actin filaments that have had helical order imposed upon them, and then allowed to relax.