Authors: Ryo Nitta, Yasushi Okada & Nobutaka Hirokawa
X-ray crystallography of kinesin enzymes in the unstable intermediate states is challenging because an uniform shape of protein is required for its crystallization. Using nucleotide analogs is one of the frequently-used techniques to solve this problem. We previously used ATP and ADP-phosphate analogs to solve the crystal structures of kinesin ATPase before and just at the point of ATP hydrolysis (1-3). However, since there are no analogs suitable for the Mg- and ADP-releasing processes, crystallization of these processes has been perceived to have many challenges and difficulties. The protocol outlined below describes how we have succeeded in crystallizing kinesin in the Mg-releasing intermediate states. Albeit this protocol will not be a panacea for all enzymes, we hope this procedure might be helpful in crystallizing some other enzymes in this transitional state.
Step 1: Protein should be pure. EDTA induces the release of magnesium and NaN3 stabilizes the EDTA-treated KIF1A.
Step 2: The concentration of KIF1A should be more than 40 mg/ml. Below this concentration, crystallization does not occur.
Step 3 to 5: Since a slow chemical reaction of Mg-release or ADP-release proceeds in crystals, crystals may appear in the hanging-drop about three days after crystallization and disappear after around seven days. The critical parameters on crystallization are protein concentration, apyrase concentration, concentration of PEG4,000 and the timing of harvesting the crystals.
Crystals in the early intermediate state of Mg-release most likely are obtained by harvesting the crystals relatively shortly after crystallization (three or four days after crystallization) from the drop including 2-fold excess apyrase. Additionally, the crystals in the late intermediate state of Mg-release may be obtained by harvesting it relatively later (six or seven days after crystallization) from the drop including 10-fold excess apyrase. The crystals in the mid-intermediate state of Mg-release may be obtained under the condition in-between.
Structural model for strain-dependent microtubule activation of Mg-ADP release from kinesin, Ryo Nitta, Yasushi Okada, and Nobutaka Hirokawa, Nature Structural & Molecular Biology 15 (10) 1067 - 1075 21/09/2008 doi:10.1038/nsmb.1487
Ryo Nitta, Yasushi Okada & Nobutaka Hirokawa, Department of Cell Biology and Anatomy, University of Tokyo, Graduate School of Medicine
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.239. Originally published online 26 November 2008.