Binding of nucleotides at the active site modulates the local and global conformation of myosin in muscle fibres

Differential scanning calorimetry and electron paramagnetic resonance exper iments were performed on glycerinated muscle fibres to study the effect of the binding of nucleotides (ADP and AMP.PNP) to myosin. The thermal unfoldi ng of muscle fibres showed three discrete domain regions with thermal stabi lities of 52.2, 58.8 and 67.8 degreesC. AMP.PNP markedly affected the trans itions, implying the strong interaction between AMP.PNP and catalytic domai n, and partial dissociation of heads from actin. ADP produced only small ch anges in transition temperatures. Spectrum deconvolution performed on isothiocyanate-labelled fibres in AMP.P NP-state resulted in two populations; 50% of labels was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly orient ed. The conformation of the myosin heads which showed high degree of order were in the strongly binding ADP-state, the heads being attached to actin d iffer from those of heads in rigor. The results support the suggestion that the attached heads in strongly binding state to actin might have different local conformations.