Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans

Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000solubilized fraction. Our enzymatic assay suggests that digitonin- or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 & DEG;C), and pH (8.0). The purified Cho1 has a Km for its substrate CDPdiacylglycerol of 72.20 & mu;M with a Vmax of 0.079 nmol/ (& mu;g*min) while exhibiting a sigmoidal kinetic curve for its other determination and small drug screening.

Automated summary

Phosphatidylserine (PS) synthase encoded by the CHO1 gene is a potential drug target for antifungals against systemic candidiasis. This study investigated methods to solubilize and purify Cho1 protein, which has a transmembrane nature. The results showed that digitonin or DDM were the most effective detergents for solubilization, and protein purification was achieved through affinity chromatography and TEV protease treatment. The purified Cho1 protein exhibited optimal enzyme activity under specific conditions of temperature, pH, and manganese and detergent concentrations, which is important for substrate determination and drug screening.