Expression, purification and crystallization of rat brain kinesin / 生物工程学报

Kinesin is a motor protein that uses the energy from ATP hydrolysis to move along the microtubule system. To investigate how the chemical energy stored in ATP is converted to mechanical movement, the corresponding N-terminal region of rat brain kinesin was expressed in BL21-Codon Plus (DE3)-RP competent cells. After SP-cation exchange chromatography and size exclusion chromatography, the protein yield reached 10 mg/L culture with the purity above 95%. The purified protein had ATPase activity and specifically reacted with the kinesin antibody in the Western blotting analysis. The purified kinesin was crystallized under the following condition: 1.7 mol/L (NH4)2SO4, 500 mmol/L NaCl, 20% glycerol. The kinesin crystal can diffract up to 2.0 angstroms resolution.

Crystallography of ATP hydrolysis mechanism in rat brain kinesin / 生物工程学报

Rat brain kinesin is a conventional kinesin that uses the energy from ATP hydrolysis to walk along the microtubule progressively. Studying how the chemical energy in ATP is utilized for mechanical movement is important to understand this moving function. The monomeric motor domain, rK354, was crystallized. An ATP analog, AMPPNP, was soaked in the active site. Comparing the complex structure of rK354 x AMPPNP and that of rK354ADP, a hypothesis is proposed that Glu237 in the Switch II region sensors the presence of gamma-phosphate and transfers the signal to the microtubule binding region.