Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli.
A truncated domain of the alpha-subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH392) extends to amino acid 392 and contains the complete N-terminal region of kinesin which is highly conserved between species. The DKH392 construct includes an additional 52 amino acids beyond the minimal motor domain of 340 amino acid residues which has been previously characterized as DKH340 (Huang, T.-G., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16493-16501). The s20,w values for DKH340 and DKH392 are 3.3 and 5.2 S and the D20,w values are 7.7 x 10(-7) and 4.9 x 10(-7) cm3 s-1, respectively. These results indicate that DKH340 is a monomer in solution, but DKH392 is a dimer. In the presence of adenosine 5-(beta,gamma-imido)triphosphate, DKH392 binds to microtubules with a stoichiometry of two head domains (one DKH392 dimer) per tubulin heterodimer in contrast to the tight binding of one DKH340 per tubulin heterodimer. Electron microscopy indicates that both DKH340 monomers and DKH392 dimers decorate microtubules with a periodicity of 8 nm.
Huang
T G
TG
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Suhan
J
J
Hackney
D D
DD
D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D004330:Drosophila / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009068:Movement; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000266:Adenylyl Imidodiphosphate; D016547:Kinesins