ATPASE KINETICS OF THE DICTYOSTELIUM-DISCOIDEUM MYOSIN-II MOTOR DOMAIN

Structural characterization of the mode of interaction of nucleotides bound to myosin has relied upon the crystal structure of the Dictyoste lium discoideum myosin II motor domain. This fragment, denoted SldC, l acks the regulatory domain and light chain subunits and may therefore fail to display the normal ATPase activity of the intact myosin molecu le. Here we show that the elementary steps of the SldC ATPase pathway and the effects of actin are similar to those of the complete myosin h ead fragment. This indicates that truncation at residue E759, with the removal of the light chain binding sites, is not crucial to catalytic activity. In particular, SldC does not show the anomolous tight bindi ng of ADP displayed by the slightly shorter M754 construct reported el sewhere. We also show that the fluorescent analogue Cy3-EDA-ATP is a g ood substrate for SldC and demonstrate the use of fluorescence correla tion spectroscopy to determine the affinity of Cy3-EDA-ADP using micro gram quantities of proteins. (C) Chapman & Hall Ltd.