DICTYOSTELIUM-DISCOIDEUM MYOSIN-II - CHARACTERIZATION OF FUNCTIONAL MYOSIN MOTOR FRAGMENTS

The transient kinetic properties of the recombinant myosin head fragme nts M761 and M781, which both lack the light chain binding domain (LCB D) and correspond to the first 761 and 781 residues of Dictyostelium d iscoideum myosin II, were compared with those of the subfragment 1-lik e fragment M864 and a shorter catalytic domain fragment M754. The prop erties of M761, M781, and M864 are almost identical in regard to nucle otide binding, nucleotide hydrolysis, actin binding, and the interacti ons between actin and nucleotide binding sites. Only the rate of the h ydrolysis step was significantly faster for M761 and the affinity of M 781 for actin significantly weaker than for M864. This indicates that the LCBD plays no major role in the biochemical behavior of the myosin head. In contrast, loss of the peptide between 754 and 761 produced s everal major changes in the property of M754 as documented previously [Woodward, S. K. A., Geeves, M. A., & Manstein, D. J. (1995) Biochemis try 34, 16056-16064]. We further show that C-terminal extension of M76 1 with one or two alpha-actinin repeats has very little effect on the behavior of the protein. The recombinant nature of M761 and the fact t hat it can be produced and purified in large amounts make it an ideal construct for systematic studies of the structure, kinetics, and funct ion of the myosin motor.