An EPR study of the rotational dynamics of actins from striated and smooth muscle and their complexes with heavy meromyosin

Abstract

The rotational motions of the actin from rabbit skeletal muscle and from chicken gizzard smooth muscle were measured by conventional and saturation transfer electron paramagnetic resonance (EPR) spectroscopy using maleimide spin‐label rigidly bound at Cys‐374. The conventional EPR spectra indicate a slight difference in the polarity of the environment of the label and in the rotational mobility of the monomeric gizzard actin compared to its skeletal muscle counterpart. These differences disappear upon polymerization. The EPR spectra of the two actins in their F form and in their complexes with heavy meromyosin (HMM) did not reveal any difference in the rotational dynamic properties that might be correlated with the known differences in the activation of myosin ATPase activity by smooth and skeletal muscle actin. Our results agree with earlier EPR studies on skeletal muscle actin in showing that polymerization stops the nanosecond rotational motion of actin monomers and that F‐actin undergoes rotational motion having an effective correlation time of the order of 0.1 ms. However, our measurements show that complete elimination of the nanosecond motions requires prolonged incubation of F‐actin, suggesting that the slow formation of interfilamental cross‐links in concentrated F‐actin solutions contributes to this process. We have also used the EPR spectroscopy to study the interaction between HMM and actin in the F and G form. Our results show that in the absence of salt one HMM molecule can cooperatively interact with eight monomers to produce a polymer which closely resembles F‐actin in its rotational mobility but differs from the complex of F‐actin with HMM. The results indicate that salt is necessary for further slowing down, in a cooperative manner, the sub‐millisecond internal motion in actin polymer and for a non‐cooperative change in the intramonomer conformation around Cys‐374 on the binding of HMM.