Renaturation of recombinant Treponema pallidum rare outer membrane protein1 into a trimeric, hydrophobic, and porin-active conformation

We have previously observed that while native Treponema pallidum rare outer membrane protein 1 (Tromp1) is hydrophobic and has porin activity, recombi nant forms of Tromp1 do not possess these properties. In this study we show that these properties are determined by conformation and can be replicated by proper renaturation of recombinant Tromp1. Native Tromp1, but not the 4 7-kDa lipoprotein, extracted from whole organisms by using Triton X-114, wa s found to lose hydrophobicity after treatment in 8 hi urea, indicating tha t Tromp1's hydrophobicity is conformation dependent. Native Tromp1 was puri fied from 0.1% Triton X-100 extracts of whole organisms by fast-performance liquid chromatography (FPLC) and shown to have porin activity in planar li pid bilayers. Cross-linking studies of purified native Tromp1 with an 11 An gstrom cross-linking agent showed oligomeric forms consistent with dimers a nd trimers. For renaturation studies of recombinant Tromp1 (rTromp1), a 31, 109-Da signal-less construct was expressed in Escherichia coli and purified by FPLC. FPLC-purified rTromp1 was denatured in 8 M urea and then renature d in the presence of 0.5% Zwittergent 3,14 during dialysis to remove the ur ea. Renatured rTromp1 was passed through a Sephacryl S-300 gel exclusion co lumn previously calibrated with known molecular weight standards. While all nonrenatured rTromp1 eluted from the column at approximately the position of the carbonic anhydrase protein standard (29 kDa), all renatured rTromp1 eluted at the position of the phosphorylase b protein standard (97 kDa), su ggesting a trimeric conformation. Trimerization was confirmed by using an 1 1 A cross-linking agent which showed both dimers and trimers similar to tha t of native Tromp1. Triton X-114 phase separations showed that all of renat ured rTromp1, but none of nonrenatured rTromp1, phase separated exclusively into the hydrophobic detergent phase, similar to native Tromp1. Circular d ichroism of nonrenatured and renatured rTromp1 showed a marked loss in alph a-helical secondary structure of renatured rTromp1 compared to the nonrenat ured form. Finally, renatured rTromp1, but not the nonrenatured form, showe d porin activity in planar liquid bilayers. These results demonstrate that proper folding of rTromp1 results in a trimeric, hydrophobic, and porin-act ive conformation similar to that of the native protein.