INTERNAL FLEXIBILITY OF CARDIAC MYOSINS

Conventional and saturation transfer electron paramagnetic resonance s pectroscopy (EPR and ST EPR) and differential scanning calorimetry (DS C) were used to study the motional dynamics and segmental flexibility of cardiac myosins. Cardiac myosins isolated from bovine and human hea rt muscle were spin-labelled with isothiocyanate- or maleimide-based p robe molecules at the reactive sulfhydryl sites (Cys-697 and Cys-707) of the motor domain. The maleimide probe molecules attached to human c ardiac myosin rotated with an effective rotational correlation time of 33 ns which was at least eight times shorter than the rotational corr elation time of the same label on skeletal myosin (260 ns). In the pre sence of MgADP and MgADP plus orthovanadate, flexibility changes in th e multisubunit structure of myosins were detected, but this did not le ad to changes of the overall rotational property of the myosin heads. Significant difference in the internal flexibility was detected on myo sin samples isolated from ischemic tissue, the rotational correlation time decreased to 25 ns. DSC measurements supported the view that addi tion of nucleotides produced additional loosening in the multisubunit structure of cardiac myosin. It is postulated that there is an intersi te communication between the nucleotide binding domain and the 20 kDa subunit where the reactive thiol sites are located.