CD spectroscopy
Briefly, DHODH protein were dialyzed against 10 mM phosphate buffer (pH
7.4) and incubated with different concentrations of PIP for 2 min at
room temperature. The circular dichroism (CD) spectra were measured by
chirascan circular dichroism spectrometer (Applied PhotoPhysics, United
Kingdom) in a quartz cell with a 0.5-cm path length.
Crystallization ofDHODH-PIP
complexes
Optimal crystals were grown by hanging drop vapor diffusion method at 20
°C as previously reported(Liu, Neidhardt, Grossman, Ocain & Clardy,
2000b).
Drops were prepared by mixing 1.0 µL of 20 mg/ml DHODH protein solution
which contained 2 mM PIP, 50 mM HEPES pH 7.7, 400 mM NaCl, 30%
Glycerol, 1 mM EDTA, 10 mM (N, N-dimethylundecylamine-N-oxide) UDAO, and
20.8 mM N, N-dimethyldecylamine- N-oxide (DDAO) with equal-volume
precipitant solution of 0.1 M acetate, pH 4.8, and 2.4-2.6 M ammonium
sulfate. The drops were incubated against 0.5 mL of 50 mM HEPES pH 7.7,
300 mM NaCl, 20% Glycerol, 1 mM EDTA, 10 mM UDAO and 20.8 mM DDAO.
Diffraction-quality crystals were appeared usually in three weeks and
reached the full size of 0.15× 0.15× 0.153 mm. Crystals were flash
frozen in liquid nitrogen for data collection.