Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface.
Kinesin, an ATP-dependent microtubule motor, can be studied in vitro in motility assays where the kinesin is nonspecifically adsorbed to a surface. However, adsorption can inactivate kinesin and may alter its reaction kinetics. We therefore prepared a biotinated kinesin derivative, K612-BIO, and characterized its activity in solution and when bound to streptavidin-coated surfaces. K612-BIO consists of the N-terminal 612 amino acids of the Drosophila kinesin alpha subunit linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotination of the protein. We expressed K612-BIO at high levels using the baculovirus expression vector system and purified it to near-homogeneity. The expressed protein is completely soluble, and > 90% is bound by streptavidin. K612-BIO steady-state ATPase kinetics (KM,ATP = 24 microM, K0.5, microtubule = 0.61 mg ml-1, Vmax = approximately 25 s-1 head-1, 25 degrees C) are similar to those reported for intact kinesin. ATPase kinetics are not affected by the addition of streptavidin. Enzyme bound to a surface coated with streptavidin drove microtubule gliding in the presence of 2 mM ATP at 750 +/- 130 nm s-1 (26 degrees C). Activity was abolished by pretreatment of the surface with biotin, indicating that the microtubule movements are due to specifically bound enzyme. Motility assays based on specific attachment of biotinated enzyme to streptavidin-coated surfaces will be useful for quantitative analysis of kinesin motility and may provide a way to detect activity in kinesin derivatives or kinesin-like proteins that have not yet been shown to move microtubules.
Berliner
E
E
Biophysics Program, Brandeis University, Waltham, Massachusetts 02254.
Mahtani
H K
HK
Karki
S
S
Chu
L F
LF
Cronan
J E
JE
Gelles
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D004330:Drosophila / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D016547:Kinesins / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D007700:Kinetics; D008870:Microtubules / Q000502:physiology*; D008969:Molecular Sequence Data; D009036:Moths; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D019809:Streptavidin; D014162:Transfection
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001710:Biotin; D000255:Adenosine Triphosphate; D019809:Streptavidin; D000251:Adenosine Triphosphatases; D016547:Kinesins