ADP-induced changes in ordering of spin-labelled myosin heads in muscle fibres.

Rotational dynamics and ordering of myosin heads in glycerinated skeletal muscle fibres were studied using an isothiocyanate-based spin label attached to the fast-reacting thiol sites of myosin and were compared with data obtained for maleimide and iodoacetamide spin labels attached to the same sites. The ordering of probe molecules on the millisecond time scale in the rigor state, at sarcomere length 2.2-2.3 +/- 0.1 microns, was static. Isothiocyanate probe molecules showed greater mobility; the segment holding the label rotated in the microsecond time range. In the saturation transfer EPR time domain, MgADP did not produce a significant change in the mobility of spin labels. The spectra of isothiocyanate spin-labelled fibres were analyzed in terms of two narrow distributions with mean angles of 75 degrees and 56 degrees. In the rigor state, the fractions represented approximately 76% and 24% of the total EPR absorbance. In the presence of MgADP, the conventional EPR spectra showed large changes in the ordering of isothiocyanate probe molecules towards a new distribution, the population with a theta value of 56% increased from 24% to 71% at the expense of the 75% population with no change in the mean angles of the distributions. In the case of maleimide and iodoacetamide spin-labelled fibres, however, the effect of MgADP on the probe angular distribution was small.