EPR evidence for nucleotide effects on attached cross-bridges

We have used electron paramagnetic resonance (EPR) to study the effects of ATP and nucleotide analogs (mainly AMPPNP) on the orientation (measured by conventional EPR) and microsecond rotational dynamics (measured by saturation transfer EPR, STEPR) of spin-labeled myosin heads, both in glycerinated muscle fibers and in solutions of purified S1 and actin. Attachment to actin was determined by stiffness measurements in fibers and by covalent cross-linking in acto-S1. Our goal is to determine whether these nucleotides induce conformations of attached cross-bridges that have head orientations or motions significantly different from rigor. While all heads are immobile and similarly oriented in fibers in rigor, relaxation by ATP produces great orientational disorder that is dynamic on the microsecond time scale. AMPPNP produces intermediate amounts of both disorder and motion. However, even at saturating levels of AMPPNP, there are two principal resolved populations of heads, whose motion and orientation are indistinguishable from those of rigor and relaxation. Thus, there is no evidence that AMPPNP induces a myosin head conformation differing in either orientation or rotational motion from the rigor and relaxed states. Since fiber stiffness is not significantly decreased by AMPPNP, even though up to 50% of myosin heads are dynamically disordered, some of the dynamically disordered heads are probably in attached cross-bridges. Simultaneous measurements of binding and orientation of labeled S1 diffused into fibers or cross-linked to actin, indicate that AMPPNP-bound heads are dynamically disordered only when detached from actin. Thus, AMPPNP detaches one of the two heads of each cross-bridge without decreasing the cross-bridge's stiffness. In contrast to AMPPNP, ATP does induce considerable microsecond rotational mobility within cross-linked acto-S1, indicating that dynamic disorder of myosin heads may occur during the attached phase of an active cross-bridge cycle. Thus we have identified two nucleotide-induced cross-bridge conformations that are rotationally different from rigor, and similar conformations may play a role in the force-generation process.