Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres

The effect of twitchin, a thick filament protein of molluscan muscles, on the actin–myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin.

► We used polarized fluorimetry to assess the conformational changes in myosin, actin and twitchin during the ATPase cycle.
► Myosin SH1 helix, actin subunit and twitchin movements were observed.
► The effects of nonphosphorylated twitchin on the actin–myosin interaction were compared with those of phosphorylated one.
► Twitchin regulates the proportion of the strong-binding sub-states in a phosphorylation-dependent manner.
► The regulation is achieved by changes in the position and mobility of actin-binding parts of twitchin.