STRUCTURAL STUDIES OF KINESIN-NUCLEOTIDE INTERMEDIATES

We have investigated the structural changes that occur in the molecula r motor kinesin during its ATPase cycle, utilizing two bacterially exp ressed constructs, The structure of both constructs has been examined as a function of the nature of the nucleotide intermediate occupying t he active site by means of sedimentation velocity, sedimentation equil ibrium, fluorescence solute quenching, fluorescence anisotropy decay, and limited proteolysis, While the molecular weight of monomeric and d imeric human kinesin constructs, as measured by sedimentation velocity and sedimentation equilibrium, and the tryptic cleavage pattern are u naffected by the nucleotide intermediate occupying the active site, si gnificant changes in the rotational correlation time of fluorescently labeled kinesin-nucleotide intermediates can be detected, These result s suggest that kinesin contains an internal ''hinge'' whose flexibilit y varies through the course of the ATPase cycle, In prehydrolytic, ''s trong'' binding states, this hinge is relatively rigid, while in posth ydrolytic, ''weak'' binding states, it is more flexible. Our results, in conjunction with anisotropy decay studies of myosin, suggest that t hese two molecular motors may share a common structural feature; viz. weak binding states are characterized by segmental flexibility, which is lost upon assumption of a strong binding conformation.