Fluorescence-based thermal shift assay
Briefly, SYPRO orange stock solution (Invitrogen, Carlsbad NY) was
diluted 1:1000 into the assay buffer (25 mM Hepes, 150 mM NaCl, pH 7.5),
and purified DHODH was added with a final concentration of 5 μM(Lu et
al., 2017). Then, the mixture (40 µL) was loaded into the 96-well
iCycler iQ PCR plate (Bio-Rad, CA, USA) with different compounds (50
μM). The plate was heated from 25 to 80 °C with a rate of 0.50 °C /min
and the fluorescence intensity was
measured
at Ex/Em=490/530 nm using a CF×96TM Real Time System (Bio-Rad, Hercules,
CA) according to the manufacturer’s instructions.
Enzymatic
kinetic analysis
Lineweaver-Burk analysis was carried out as described previously(McLean,
Neidhardt, Grossman & Hedstrom, 2001). Briefly, a variety of
concentrations of CoQ0 (25-200 µM), DHO (25-200 µM) and
compound (1-3 µM) were incubated with DHODH (20 nM) in reaction buffer
(50 mM Tris, pH 8.0, and 150 mM KCl). The enzymatic reaction was
detected at 287 nm using a Hitachi U-2000 spectrophotometer (Tokyo,
Japan). In addition, kinetic curves of DHODH activity in the presence of
varied concentrations of PIP was generated to calculate the
Kon and Koff values. All experiments
were performed at 25 °C. The experiments were performed at least three
times.
Isothermal
titration calorimetry (ITC)
Thermodynamic characterization of DHODH-PIP interactions was performed
using an iTC200 instrument (Microcal, GE Healthcare, PA, USA) as
described before2,(Zhao et al., 2015). Before
titrations, the stock solutions of PIP and DHODH protein were diluted
with ITC buffer (50 mM HEPES, 300 mM NaCl, 10% Glycerol, 0.1% Triton)
at the concentrations of 100 µM and 20 µM, respectively. All titrations
of PIP into DHODH were performed by 19 identical injections of 2 µl with
a duration of 4 seconds per injection spaced at interval of 120 seconds
between injections following an initial injection of 0.4 µl at 25 °C.
The reaction heat of injecting PIP into the DHODH were obtained and the
data was processed by the supplied MicroCal Origin software package (PA,
USA). The final concentration of DMSO in the assay buffer is less than
3%. All the calorimetric data were calculated with the formula
ΔG
= ΔH − TΔS = −RT lnK, where T is experimental temperature, R is gas
constant, K is binding constant, ΔG represents changes of free energy,
ΔH represents changes of enthalpy and ΔS represents changes of entropy
of, respectively.