Isothermal titration calorimetry

The binding assay was performed in the microcalorimeter as previously described [26]. Briefly, lipid binding to the recombinant RpACBP-5 was analyzed in a VP-ITC microcalorimeter (MicroCal, Boston, MA, USA) equilibrated at 27°C. The protein solution (25 μM in 25 mM ammonium acetate buffer, pH 6.0) was loaded into the calorimeter cell and titrated with 0.5 M lauroyl-CoA in the same buffer, using 30 consecutive aliquots of 4 μl at 3 min interval. The solution was mixed at 400 rpm. The obtained data were analyzed by Origin software (MicroCal).