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  • 1 University of Pécs Biophysical Department, Faculty of Medicine H-7624 Pécs Szigeti str. 12 Hungary
  • 2 University of Pécs Central Research Laboratory, Faculty of Medicine H-7624 Pécs Szigeti str. 12 Hungary
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Abstract  

The effect of AMP.PNP on the thermal stability and dynamics of myosin head were investigated by using DSC and different spin label technique for chemically skinned muscle fibres prepared from rabbit. The thermal unfolding of the fibres in rigor, strong as well as weak-binding state showed a complex process characterizing at least three discrete domain regions with different stability (Tm =54, 58.4 and 62.3°C). The unfolding at 54°C refers to the catalytic domain of myosin, whereas transition at Tm =58.4°C represents the rod-like region. In the presence of AMP.PNP only the parameters of the last transition changed significantly (Tm =70.4°C) showing an increased interaction between actin and myosin heads being attached to actin. Measurements on MSL-fibres (labelled at Cys-707 of myosin) in the presence of AMP.PNP showed that about half of the cross-bridges dissociated from actin. This fraction had a dynamic disorder, the other population had the same spectral feature as in rigor. In contrast, on TCSL-fibres AMP.PNP increased the orientational disorder of myosin heads, a random population of spin labels was superimposed on the ADP-like spectrum showing conformational and motional changes in the internal structure of myosin heads. ST EPR measurements reported increased rotational mobility of spin labels in the presence of AMP.PNP. The DSC and EPR results suggest that in the presence of AMP.PNP the attached heads have the same global orientation as in rigor, but the internal structure undergoes a local conformational change.

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