Figure 6.

Recombinant UbiU_Pa and UbiT_Pa bind UQ₈. A, Elution profile of UbiU_Pa. 70 mg of protein was loaded on a Superdex 200 16/60 chromatography column. Quantification of UQ₈ and DMQ₈ in each fraction was performed by HPLC-ECD MS. Recovery of 73 and 76%, respectively, for UQ₈ and DMQ₈ was calculated from the total content of all fractions compared with content of the UbiU_Pa-purified fraction deposited in the Superdex 200 column. B, Fractions 10–44, analyzed by SDS-PAGE for purity. C, Elution profile of UbiT_Pa on a Superdex 200 16/60 column. Inset, SDS-PAGE. Lane 1, 32-kDa TrxA-UbiT_Pa fusion protein; lane 2, after digestion with thrombin (UbiT_Pa, 19.6 kDa; TrxA, 12.1 kDa); lane 3, pooled fractions 20–30 of UbiT_Pa. Quantification of UQ₈ (pool of fractions 20–30) was performed by HPLC-ECD MS. D, Protein-lipid overlay assay between UbiU_Pa and UbiT_Pa and different lipid ligands. 2 μl of six different lipid/compound potential candidates (1, UQ₈; 2, UQ₁₀; 3, solanesol; 4, 3-methylcatechol; 5, cholesterol; 6, POPE) at 20 mm final concentration were spotted on a PVDF membrane and then incubated with UbiT_Pa or UbiU_Pa (both proteins at 0.2 µg/ml final concentration). Detection of bound proteins was performed by chemiluminescence, as described in Experimental procedures. MW, molecular weights.