Rotational dynamics of spin-labelled muscle proteins.

Electron paramagnetic resonance (EPR) spectroscopy on spin-labelled proteins was used to investigate the role of rotational motions in the two key ATPase systems of muscle: (a) the force-generating interaction of myosin and actin and (b) the calcium pump of sarcoplasmic reticulum (SR). The spin labels in these studies were rigidly fixed to the protein framework, so there were none of the nanosecond motions detectable by conventional EPR techniques. Thus conventional EPR was useful only in the study of static orientation in oriented systems (parallel muscle fibres or membrane bilayers), while saturation transfer EPR was required to detect protein motions in the microsecond range. Spin labels attached to myosin 'heads' were used to test the proposal that these heads form force-generating cross-bridges to actin that rotate when driven by the ATPase cycle. These probes are highly oriented and strongly immobilized when heads are bound to actin, but undergo microsecond rotations when the heads are detached from actin. During contraction, the majority of heads are mobile and disoriented, indicating detachment, while the remainder appear to be attached with the same orientation as in the absence of ATP. Thus the probed part of the myosin head does not appear to rotate during force generation. Saturation transfer EPR was used to monitor the microsecond rotational motions of the Ca2+-ATPase in SR membranes, while conventional EPR was used to monitor lipid hydrocarbon rotations in the microsecond time range. Changes in lipid fluidity and protein mobility, induced by changes in lipid/protein ratio, lipid composition, [Ca2+] and protein cross-linking, correlate well with changes in enzymic activity, suggesting the importance of molecular motions for Ca2+ transport.