Structural and functional features of one- and two-headed biotinated kinesin derivatives.
The oligomeric structure was determined for four recombinant kinesin derivatives containing N-terminal fragments of the kinesin alpha-subunit. Some of the proteins were dimeric (two-headed) molecules with mechanochemical properties similar to those of intact kinesin. Comparison of the primary and quaternary structures of the derivatives with those of intact kinesin suggests that structures distinct from the long alpha-helical coiled-coil rod domain contribute to subunit self-association. Three of the proteins contain a single engineered site for post-translational biotination in vivo; this facilitates analysis of motility in experiments in which the proteins are specifically bound to streptavidin-conjugated microscopic plastic beads. One of the derivatives is monomeric (one-headed); like the two-headed derivatives, it is functional in the motility assay and is a microtubule-dependent ATPase. Unlike 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 ability of molecules with a one-headed solution structure to generate movement is consistent with a translocation-generating conformational change internal to the kinesin head. A simple set of coupling rules can be used to formulate consistent mechano-chemical mechanisms that explain movement by both one- and two-headed kinesin molecules.
Gelles
J
J
Department of Biochemistry, Center for Complex Systems, Waltham, Massachusetts 02254, USA.
Berliner
E
E
Young
E C
EC
Mahtani
H K
HK
Perez-Ramirez
B
B
Anderson
K
K
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001696:Biomechanical Phenomena; D055592:Biophysical Phenomena; D001703:Biophysics; D001710:Biotin / Q000737:chemistry; D004330:Drosophila; D066298:In Vitro Techniques; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008870:Microtubules / Q000502:physiology; D008954:Models, Biological; D015394:Molecular Structure; D009068:Movement / Q000502:physiology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D001710:Biotin; D000255:Adenosine Triphosphate; D016547:Kinesins