STRUCTURAL AND FUNCTIONAL FEATURES OF ONE-HEADED AND 2-HEADED BIOTINATED KINESIN DERIVATIVES

The oligomeric structure was determined for four recombinant kinesin d erivatives containing N-terminal fragments of the kinesin alpha-subuni t. Some of the proteins were dimeric (two-headed) molecules with mecha nochemical properties similar to those of intact kinesin. Comparison o f the primary and quaternary structures of the derivatives with those of intact kinesin suggests that structures distinct from the long alph a-helical coiled-coil rod domain contribute to subunit self-associatio n. Three of the proteins contain a single engineered site for post-tra nslational biotination in vivo; this facilitates analysis of motility in experiments in which the proteins are specifically bound to strepta vidin-conjugated microscopic plastic beads. One of the derivatives is monomeric (one-headed); like the two-headed derivatives, it is functio nal in the motility assay and is a microtubule-dependent ATPase. Unlik e intact kinesin and the two-headed derivatives, the one-headed enzyme fails to track microtubule protofilaments. This confirms a prediction of proposed ''hand-over-hand'' mechanisms of kinesin movement. The ab ility of molecules with a one-headed solution structure to generate mo vement is consistent with a translocation-generating conformational ch ange internal to the kinesin head. A simple set of coupling rules can be used to formulate consistent mechanochemical mechanisms that explai n movement by both one- and two-headed kinesin molecules.