BtubA-BtubB heterodimer is an essential intermediate in protofilament assembly.

Background: BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to alpha-tubulin and BtubA to beta-tubulin. A 1:1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles.

In vitro assembly and GTP hydrolysis by bacterial tubulins BtubA and BtubB.

Arecent study identified genuine tubulin proteins, BtubA and BtubB, in the bacterial genus Prosthecobacter. We have expressed BtubA and BtubB in Escherichia coli and studied their in vitro assembly. BtubB by itself formed rings with an outer diameter of 35-36 nm in the presence of GTP or GDP.

Mechanistic analysis of the Saccharomyces cerevisiae kinesin Kar3.

Kar3 is a minus-end-directed microtubule motor that is implicated in meiotic and mitotic spindle function in Saccharomyces cerevisiae. To date, the only truncated protein of Kar3 that has been reported to promote unidirectional movement in vitro is GSTKar3. This motor contains an NH2-terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended alpha-helical coiled-coil.

Mechanistic analysis of the mitotic kinesin Eg5.

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k(cat) = 5.