title: Renaturation of recombinant Treponema pallidum rare outer membrane protein 1 into a trimeric, hydrophobic, and porin-active conformation.
abstract: We have previously observed that while native Treponema pallidum rare outer membrane protein 1 (Tromp1) is hydrophobic and has porin activity, recombinant 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 47-kDa lipoprotein, extracted from whole organisms by using Triton X-114, was found to lose hydrophobicity after treatment in 8 M urea, indicating that Tromp1's hydrophobicity is conformation dependent. Native Tromp1 was purified from 0.1% Triton X-100 extracts of whole organisms by fast-performance liquid chromatography (FPLC) and shown to have porin activity in planar lipid bilayers. Cross-linking studies of purified native Tromp1 with an 11 A cross-linking agent showed oligomeric forms consistent with dimers and 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 renatured in the presence of 0.5% Zwittergent 3,14 during dialysis to remove the urea. Renatured rTromp1 was passed through a Sephacryl S-300 gel exclusion column 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), suggesting a trimeric conformation. Trimerization was confirmed by using an 11 A cross-linking agent which showed both dimers and trimers similar to that of native Tromp1. Triton X-114 phase separations showed that all of renatured rTromp1, but none of nonrenatured rTromp1, phase separated exclusively into the hydrophobic detergent phase, similar to native Tromp1. Circular dichroism of nonrenatured and renatured rTromp1 showed a marked loss in alpha-helical secondary structure of renatured rTromp1 compared to the nonrenatured form. Finally, renatured rTromp1, but not the nonrenatured form, showed porin activity in planar liquid bilayers. These results demonstrate that proper folding of rTromp1 results in a trimeric, hydrophobic, and porin-active conformation similar to that of the native protein.
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, School of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA.
authors.forename: H H
authors.initials: HH
authors.lastname: Zhang
authors.forename: D R
authors.initials: DR
authors.lastname: Blanco
authors.forename: M M
authors.initials: MM
authors.lastname: Exner
authors.forename: E S
authors.initials: ES
authors.lastname: Shang
authors.forename: C I
authors.initials: CI
authors.lastname: Champion
authors.forename: M L
authors.initials: ML
authors.lastname: Phillips
authors.forename: J N
authors.initials: JN
authors.lastname: Miller
authors.forename: M A
authors.initials: MA
authors.lastname: Lovett
mesh_terms: D001426:Bacterial Proteins; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003902:Detergents / Q000494:pharmacology; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D018272:Porins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D020673:Protein Renaturation / Q000187:drug effects; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013388:Succinimides / Q000494:pharmacology; D014210:Treponema pallidum / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D018272:Porins; D011994:Recombinant Proteins; D013388:Succinimides; C093836:Tromp1 protein, Treponema pallidum; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; C035760:bis(sulfosuccinimidyl)suberate