FIG. 1.

Pgp3 purified from an E. coli expression system is trimeric. (A) Pgp3 was purified from E. coli expressing a GST-Pgp3 fusion protein via affinity binding to glutathione agarose beads and cleaving of the fusion tag GST. The purified Pgp3 was either left untreated (lane 1) or treated with 2% SDS plus 5 min of boiling (lane 2), and the samples were subjected to electrophoresis in a 12% polyacrylamide native gel. The resolved protein bands were visualized by staining with Coomassie blue dye. Note that the untreated Pgp3 migrated at the ∼84 kDa position (corresponding to the molecular mass of trimeric Pgp3) whereas the SDS-denatured Pgp3 migrated at the ∼28 kDa position (corresponding to the monomeric form of Pgp3), as indicated on the right of the gel image. (B) Sedimentation velocity analyses of Pgp3 at concentrations of 0.6 (panel a), 1 (panel b), and 1.5 (panel c) mg/ml. The G(s) plot was obtained using the van Holde and Weischet method (44). Note that, regardless of the protein concentration, most readings (open circles) formed a vertical line with an S₂₀,w value of ∼4.2 (x axis). Assuming that Pgp3 maintains a similar overall shape, the theoretic S₂₀,w values for the trimeric, dimeric, and monomeric forms of Pgp3 are expected to be 4.2 (triple arrows on the top of the figure), 3.7 (double arrows), and 2.3 (single arrow), respectively. (C) Monte Carlo analysis of the sedimentation velocity of Pgp3 revealed a molecular mass of 84 kDa, corresponding to the molecular mass of a trimeric Pgp3, and a frictional ratio of 1.6, suggesting that the Pgp3 trimers were in an elongated form. (D) The stability of Pgp3 trimers was analyzed by treating Pgp3 with various combinations of denaturing agents under various conditions as indicated on top of the figure. The mobility of the treated Pgp3 molecules was monitored using a 12% native gel plus a Western blot assay with an anti-Pgp3 mouse polyclonal antibody known to recognize both native and denatured Pgp3. Note that neither of the reducing agents (2% 2-ME or 20 mM DTT, for 30 min of exposure) altered the ratio of the trimeric versus monomeric Pgp3 molecules (lanes 2 and 3). Treatment with 8 M urea and 2% SDS for 30 min converted ∼50% of the Pgp3 into monomers (lanes 4 and 5). Inclusion of DTT in the SDS treatment did not further increase the monomer/trimer ratio (lane 6). Boiling for 1 min caused aggregation of Pgp3, and most Pgp3 molecules stayed in the stacking gel (lane 7). Heating for 5 min in the presence of SDS completely converted all Pgp3 trimers into monomers (lane 9).