DSC and EPR study on AMP.PNP, BeFx and AlF4 containing myosin nucleotide complexes

Abstract

Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated skeletal muscle fibres to study the effect of the binding of nucleotides and nucleotide analogues to myosin. The thermal unfolding of muscle fibres in rigor showed three discrete domain regions with thermal stability of 52.2, 58.8 and 67.8°C. AMP.PNP and ATP plus AlF3 or BeF2 affected markedly the transitions, which implies the strong interaction between AMP.PNP or nucleotide analogues and catalytic domain of myosin, and a partial dissociation of heads from actin. ADP.BeFx and states model the transition states of the ATP hydrolysis cycle which precede the powerstroke of the muscle fibres. Spectrum deconvolution on isothiocyanate-labelled fibres in AMP.PNP-state resulted in two populations; 50% of labels was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The myosin heads which showed high degree of order were in the strongly binding ADP-state. The spectra in - and ADP.BeFx state reflected random orientation of labels with increased rotational mobility in comparison with rigor. The results suggest that myosin in muscle fibres in ADP.BeFx state exists in two forms.