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• W2021488049 abstract "Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a β-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition. Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a β-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition. Targeting and translocation of soluble and integral membrane proteins to the ad hoc subcellular compartments are essential for cell function and organelle biogenesis. Transport of proteins across membranes and their insertion into membranes are typically mediated by proteinaceous transmembrane complexes, and some of these pathways have been conserved throughout evolution (1Alder N.N. Theg S.M. Trends Biochem. Sci. 2003; 28: 442-451Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). With their complex envelope structure, Gram-negative bacteria and eukaryotic organelles face similar challenges for the translocation of proteins (2Matouschek A. Glick B.S. Nat. Struct. Biol. 2001; 8: 284-286Crossref PubMed Scopus (26) Google Scholar, 3Herrmann J.M. Trends Microbiol. 2003; 11: 74-79Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 4Reumann S. Keegstra K. Trends Plant Sci. 1999; 4: 302-307Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In the outer membrane of Gram-negative bacteria, specific membrane-embedded β-barrel proteins are essential components of protein transport machineries (5Thanassi D.G. Hultgren S.J. Curr. Opin. Cell Biol. 2000; 12: 420-430Crossref PubMed Scopus (238) Google Scholar). Similarly, polypeptide-transporting β-barrel proteins are also found in the outer membranes of eukaryotic organelles of prokaryotic origin such as chloroplasts and mitochondria (6Schnell D.J. Kessler F. Blobel G. Science. 1994; 266: 1007-1012Crossref PubMed Scopus (331) Google Scholar, 7Wimley W.C. Curr. Opin. Struct. Biol. 2003; 13: 404-411Crossref PubMed Scopus (345) Google Scholar). The recently revealed Omp85/TpsB superfamily of outer membrane proteins is dedicated to protein transport in most major kingdoms of life, although no members have been identified in Archaebacteria yet (8Moslavac S. Mirus O. Bredemeier R. Soll J. von Haeseler A. Schleiff E. FEBS J. 2005; 272: 1367-1378Crossref PubMed Scopus (73) Google Scholar, 9Yen M.-R. Peabody C.R. Partovi S.M. Zhai Y. Tseng Y.-H. Saier M.H. Biochim. Biophys. Acta. 2002; 1562: 6-31Crossref PubMed Scopus (155) Google Scholar). TpsB transporters are found in two-partner secretion systems, developed by Gram-negative bacteria for the secretion of large “TpsA” proteins destined to the cell surface or the milieu and serving mostly as virulence factors (10Jacob-Dubuisson F. Fernandez R. Coutte L. Biochim. Biophys. Acta. 2004; 1694: 235-257Crossref PubMed Scopus (138) Google Scholar). The other members of the Omp85/TpsB superfamily are transporter proteins included in large hetero-oligomeric complexes, such as the Toc75 homologs in chloroplasts, the Tob55/Sam50 homologs in mitochondria and the Omp85 homologs in Gram-negative bacteria. These transporters mediate inward protein transport across the chloroplast outer membrane and assembly of β-barrel proteins into the outer membrane of mitochondria and Gram-negative bacteria, respectively (11Schleiff E. Soll J. Kuchler M. Kuhlbrandt W. Harrer R. J. Cell Biol. 2003; 160: 541-551Crossref PubMed Scopus (178) Google Scholar, 12Paschen S.A. Waizenegger T. Stan T. Preuss M. Cyrklaff M. Hell K. Rapaport D. Neupert W. Nature. 2003; 426: 862-866Crossref PubMed Scopus (347) Google Scholar, 13Kozjak V. Wiedemann N. Milenkovic D. Lohaus C. Meyer H.E. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2003; 278: 48520-48523Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 14Voulhoux R. Bos M.P. Geursten J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (586) Google Scholar, 15Gentle I. Gabriel K. Beech P. Waller R. Lithgow T. J. Cell Biol. 2004; 164: 19-24Crossref PubMed Scopus (314) Google Scholar). Although their sizes and origins vary, the proteins of the TpsB/Omp85 superfamily are phylogenetically related and have been postulated to derive from a common ancestor, probably a simple prokaryotic channel (4Reumann S. Keegstra K. Trends Plant Sci. 1999; 4: 302-307Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 16Bolter B. Soll J. Schulz A. Hinnah S. Wagner R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15831-15836Crossref PubMed Scopus (139) Google Scholar, 17Eckart K. Eichacker L. Sohrt K. Schleiff E. Heins L. Soll J. EMBO Rep. 2002; 3: 557-562Crossref PubMed Scopus (95) Google Scholar). They are all predicted to be composed of a number of amphipathic β-strands, most likely forming a transmembrane β-barrel, and channel activity was demonstrated for several of them (12Paschen S.A. Waizenegger T. Stan T. Preuss M. Cyrklaff M. Hell K. Rapaport D. Neupert W. Nature. 2003; 426: 862-866Crossref PubMed Scopus (347) Google Scholar, 16Bolter B. Soll J. Schulz A. Hinnah S. Wagner R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15831-15836Crossref PubMed Scopus (139) Google Scholar, 18Könninger U.W. Hobbie S. Benz R. Braun V. Mol. Microbiol. 1999; 32: 1212-1225Crossref PubMed Scopus (59) Google Scholar, 19Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (189) Google Scholar, 20Hinnah S.C. Wagner R. Sveshnikova N. Harrer R. Soll J. Biophys. J. 2002; 83: 899-911Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 21Jacob-Dubuisson F. El-Hamel C. Saint N. Guedin S. Willery E. Molle G. Locht C. J. Biol. Chem. 1999; 274: 37731-37735Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Their sequences share conserved C-proximal sequence motifs of unknown function (8Moslavac S. Mirus O. Bredemeier R. Soll J. von Haeseler A. Schleiff E. FEBS J. 2005; 272: 1367-1378Crossref PubMed Scopus (73) Google Scholar, 9Yen M.-R. Peabody C.R. Partovi S.M. Zhai Y. Tseng Y.-H. Saier M.H. Biochim. Biophys. Acta. 2002; 1562: 6-31Crossref PubMed Scopus (155) Google Scholar, 17Eckart K. Eichacker L. Sohrt K. Schleiff E. Heins L. Soll J. EMBO Rep. 2002; 3: 557-562Crossref PubMed Scopus (95) Google Scholar). Despite their implication in critical biological processes such as membrane biogenesis and secretion of virulence factors, none of these proteins has been characterized structurally so far, and the currently available topology models are somewhat conflicting (13Kozjak V. Wiedemann N. Milenkovic D. Lohaus C. Meyer H.E. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2003; 278: 48520-48523Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 14Voulhoux R. Bos M.P. Geursten J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (586) Google Scholar, 15Gentle I. Gabriel K. Beech P. Waller R. Lithgow T. J. Cell Biol. 2004; 164: 19-24Crossref PubMed Scopus (314) Google Scholar, 18Könninger U.W. Hobbie S. Benz R. Braun V. Mol. Microbiol. 1999; 32: 1212-1225Crossref PubMed Scopus (59) Google Scholar, 22Sveshnikova N. Grimm R. Soll J. Schleiff E. Biol. Chem. 2000; 381: 687-693Crossref PubMed Scopus (61) Google Scholar, 23St. Geme III, J.W. Grass S. Mol. Microbiol. 1998; 27: 617-630Crossref PubMed Scopus (95) Google Scholar, 24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Bordetella pertussis secretes its major adhesin, FHA, via a TpsB family member, the outer membrane transporter FhaC (25Jacob-Dubuisson F. Locht C. Antoine R. Mol. Microbiol. 2001; 40: 306-313Crossref PubMed Scopus (212) Google Scholar). A topology model for FhaC was proposed earlier, based on sequence alignments of several TpsB proteins, predicting 19 amphipathic anti-parallel β-strands linked by large surface loops and periplasmic turns (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The C-terminal portion of this model was reasonably well validated by an epitope insertion method, whereas the N-terminal moiety has remained significantly less well defined (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). FhaC was shown to form ion-permeable channels in artificial membranes, although the relationship between this pore activity and the FHA-secreting activity of FhaC has not been established (21Jacob-Dubuisson F. El-Hamel C. Saint N. Guedin S. Willery E. Molle G. Locht C. J. Biol. Chem. 1999; 274: 37731-37735Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). A collection of mutant FhaC proteins each carrying a two-residue insertion at a specific sequence position were generated previously (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In this work, a number of these mutant FhaCs with varied FHA secretion activities were purified, and their electrophysiological characteristics were studied at both macroscopic and single-channel levels and compared with those of the wild type (WT) 6The abbreviations used are: WTwild typeTEVtobacco etch virus. protein. We have thus established that the C-terminal two-thirds of FhaC participate in channel formation, and its first 200 residues most likely form a functionally distinct domain. The results also suggest that the ion channel corresponds to the protein-conducting pore. wild type tobacco etch virus. Plasmid Construction—pFJD118 was constructed as follows. pFcc3 (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) was linearized by restriction with BamHI, which cleaves at a unique site positioned after the third codon into the sequence of mature FhaC. The oligonucleotides 5′-GATCCCACCATCACCACCATCACGGGCCCG-3′ and 5′-GATCCGGGCCCGTGATGGTGGTGATGGTGG-3′ were annealed and inserted into BamHI-restricted pFcc3. The orientation of that linker was verified by sequencing, and the resulting plasmid was called pFcc3-His6. The latter plasmid was restricted with XbaI and SacI, and the fhaC-containing DNA fragment was inserted into the same sites of pET24d (Novagen), resulting in pFJD118. To prepare the pFJD118 derivatives harboring fhaC each with a given insertion, pFJD118 was restricted with PstI (which cleaves a few nucleotides 3′ of the position of the BamHI site in fhaC) and SacI (which cleaves after the termination codon of fhaC). The PstI-SacI fragments prepared from the corresponding pFccx, pMycx, or pTEVx plasmids (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), each coding for FhaC with a particular insertion (or deletion), were exchanged for the WT fragment of pFJD118. The resulting plasmids were called pT7FcxB (where x represents the position of the codon with a BamHI insert), pT7FcxM (for a Myc-encoding insert at codon x), pT7FcxT (for a tobacco etch virus (TEV) cleavage site encoding insert at codon x), or pT7FcΔx-y (with a deletion between codons x and y). The corresponding amino acid sequences of the mutants are shown in the supplemental Table. To construct pFJD150, the plasmid encoding the C-terminal truncate FhaC-Δ3–206, pFcc206 was restricted with BamHI and HindIII, and the resulting fragment coding for the last 349 residues of FhaC was inserted into a pFJD118 derivative called pFJD138 in replacement of its own BamHI-HindIII fragment. pFJD138 is similar to pFJD118 except that there is a BamHI site only 3′ of the His6 tag-coding linker, but not in the 5′ position. The resulting pFJD150 encodes the C-terminal truncate of FhaC with a His6 sequence located three residues after the signal peptide cleavage site. Expression and Purification of Wild Type and Mutant FhaC Proteins—The WT and mutant FhaC proteins were produced and purified, as described previously (21Jacob-Dubuisson F. El-Hamel C. Saint N. Guedin S. Willery E. Molle G. Locht C. J. Biol. Chem. 1999; 274: 37731-37735Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) from Escherichia coli BL21(DE3)-omp5 (26Prilipov A. Phale P.S. van Gelder P. Rosenbusch J.P. Koebnik R. FEMS Microbiol. Lett. 1998; 163: 65-72Crossref PubMed Google Scholar) carrying each of the pT7Fc plasmids, with the following modifications. After harvesting the membrane fractions by ultracentrifugation, two steps of extraction were performed successively with 0.8 and 1.5% β-octyl glucoside. The second detergent extract was chromatographed as described previously (21Jacob-Dubuisson F. El-Hamel C. Saint N. Guedin S. Willery E. Molle G. Locht C. J. Biol. Chem. 1999; 274: 37731-37735Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), except that 1% Elugent was used in the buffers rather than 0.4%. The metal-chelate chromatography was performed with 400 mm imidazole in the elution buffer. Secretion Assay—E. coli UT5600 bacteria harboring pFJD12 (coding for Fha44, a C-terminally truncated version of FHA whose secretion properties parallel those of FHA (27Jacob-Dubuisson F. Buisine C. Mielcarek N. Clement E. Menozzi F.D. Locht C. Mol. Microbiol. 1996; 19: 65-78Crossref PubMed Scopus (49) Google Scholar)) and each of the plasmids of the pFcx series (or pTEVx or pMycx) were grown as described previously (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 28Guédin S. Willery E. Locht C. Jacob-Dubuisson F. Mol. Microbiol. 1998; 29: 763-774Crossref PubMed Scopus (59) Google Scholar). The culture supernatants were harvested following a 2-h period of induction with 1 mm isopropyl 1-thio-β-d-galactopyranoside and were subjected to electrophoresis in polyacrylamide gels in the presence of SDS. The proteins were then transferred onto a nitrocellulose membrane, and immunodetection was performed using a mixture of two monoclonal antibodies raised against FHA, F4 and F5 (29Coutte L. Antoine R. Drobecq H. Locht C. Jacob-Dubuisson F. EMBO J. 2001; 20: 5040-5048Crossref PubMed Scopus (118) Google Scholar). To compare the amounts of FhaC produced by those strains, bacteria were harvested from liquid cultures at mid-exponential phase, washed in 20 mm Hepes (pH 7), and resuspended in 1/10 volume of the same buffer. Equal amounts of bacteria were used for all strains. They were broken by passages in a French press, and the clarified lysates were subjected to a 1-h ultracentrifugation at 100,000 × g to pellet the membranes. The pellets were resuspended in 20 mm Hepes (pH 7), and membrane aliquots from each strain were analyzed by SDS-PAGE and immunoblotting using anti-FhaC antibodies (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The amounts of Fha44 and FhaC in all strains were determined by densitometry scanning of immunoblots. The secretion activities were calculated by determining the ratios Fha44/FhaC and setting the secretion activity of the strain producing WT FhaC to 100%. Planar Lipid Bilayer Recordings—From a 0.5% solution of azolectin (Sigma) in hexane, virtually solvent-free planar lipid bilayers were formed by the apposition of two monolayers as described (21Jacob-Dubuisson F. El-Hamel C. Saint N. Guedin S. Willery E. Molle G. Locht C. J. Biol. Chem. 1999; 274: 37731-37735Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The membrane was formed over a 125–200-μm diameter hole in a thin Teflon film (10-μm) sandwiched between two glass cells pretreated with hexadecane/hexane (1:40, v/v). The cis- and trans-compartments contained 1 m KCl buffered with 10 mm Hepes (pH 7.4). 0.5–5 μl of purified FhaC (initial concentration of 0.2–0.5 mg/ml) were diluted 100-fold in 1% octyl-polyoxyethylene and added to the cis-compartment. The measuring cell was connected with an Ag/AgCl electrode at the cis-side, and the electrode on the so-called trans-side was grounded. All experiments were performed at room temperature. In macroscopic conductance experiments, the doped membranes were subjected to slow voltage ramps (10 mV/s), and transmembrane currents were fed into an amplifier (BBA-01, Eastern Scientific, Rockville, MD). Current-voltage curves were analyzed with the SCOPE software (Bio-Logic, Claix, France). In single-channel recordings, currents were amplified and potentials were applied simultaneously by a patch clamp amplifier (RK 300, Bio-Logic). Single-channel currents were monitored using an oscilloscope (TDS 3012, Tektronix, Beaverton, OR) and stored on a CD recorder (DRA 200, Bio-Logic) for off-line analysis. CD data were then analyzed by the WinEDR (Bio-Logic) and Clampfit (Axon Instruments) software. Data were filtered at 1 kHz before digitizing at 11.2 kHz for analysis. TEV Protease Digestion of FhaC-150T Mutant—Purified FhaC-150T diluted in 0.05% dodecyl maltoside was treated with TEV protease (AcTEV Protease, Invitrogen) in 40 samples of 20 μl each. The digestion was performed overnight at 30 °C in 50 mm Tris-HCl (pH 8.0) 0.5 mm EDTA, 1 mm dithiothreitol, with 6 units of AcTEV protease (10 units/μl) in each reaction tube. The ratio of TEV protease to FhaC-150T was ∼1:1. The samples were analyzed by SDS-PAGE. Choice of the Mutant FhaC Derivative—To study the relationship between the channel properties of FhaC and its secretion activity, 20 different mutants were selected from a collection generated previously by random insertion of 6-mer BamHI linkers into the fhaC sequence (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The corresponding mutant FhaCs contain each a 2-residue insertion at a given position in the protein (Fig. 1A and supplemental Table). Four FhaC derivatives with longer c-Myc insertions (16 residues), FhaC-125M, FhaC-260M, FhaC-434 M, and FhaC-503M, were included in the analysis, as well as two additional FhaC variants, FhaC-Δ3–26 and FhaC-Δ221–228, each carrying a small deletion in predicted loops L1 and L3, respectively (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). All the mutant genes were coexpressed with the fha44 gene in E. coli UT5600 to determine the secretion activities of the corresponding FhaC proteins, as described previously (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Fha44 is an N-terminal fragment of FHA whose secretion properties parallel those of the full-length protein (27Jacob-Dubuisson F. Buisine C. Mielcarek N. Clement E. Menozzi F.D. Locht C. Mol. Microbiol. 1996; 19: 65-78Crossref PubMed Scopus (49) Google Scholar). The levels of production of the FhaC mutant proteins were determined by immunoblot analyses of total membrane fractions of the recombinant strains using anti-FhaC antibodies (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) (Fig. 1B). In parallel, we assessed Fha44 secretion by immunoblot analyses of nonconcentrated culture supernatants of these recombinant strains using anti-FHA antibodies (Fig. 1B). The secretion activities were obtained by calculating the Fha44/FhaC ratio for each strain (Table 1). The FhaC mutant proteins secreted Fha44 with widely varied efficiencies (Table 1). Altogether, the levels of Fha44 secretion were found to depend essentially on the positions of the mutations rather than on the abundance of the mutant FhaC proteins, as observed earlier (24Guédin S. Willery E. Tommassen J. Fort E. Drobecq H. Locht C. Jacob-Dubuisson F. J. Biol. Chem. 2000; 275: 30202-30210Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) (Fig. 1B and Table 1). Of note, the c-Myc insertion immediately after residue 125, although predicted to interrupt a transmembrane strand, did not affect the production of FhaC or its ability to secrete Fha44 in vivo (Fig. 1B).TABLE 1Secretion activities and electrophysiological properties of FhaC derivativesMutationSecretion activity of mutantaSecretion activities correspond to the levels of Fha44 secreted in the supernatants corrected by the levels of FhaC produced by the recombinant strains. They were obtained by determining the ratios of the intensities of the Fha44 and FhaC bands on immunoblots, with the secretion activity of the strain producing WT FhaC set to 100%. The activities shown in the table correspond to a representative experiment. The mutant FhaCs were sorted in decreasing order of activities.Channel propertiesMacroscopic I/V curveConductancebConductances were determined in 1 m KCl using single channel recordings. NC indicates not calculated because signals were too noisy; ND indicates not determined; NA indicates no activity.pS150B117WT-like1180125M109WT-likeND532B100WT-like1230Δ3-2698WT-like800-1000Δ221-22886Affected850367B81WT-like105093B77WT-like1240503M76WT-likeND395B59WT-like1115108B59Affected930260M32AffectedND462B3.3AffectedNC342B2.6WT-like1260434B1.5AffectedNC243B0Affected900-3000307B0Affected350434M0NANA495B0AffectedNCΔ3-206NDAffected200-900a Secretion activities correspond to the levels of Fha44 secreted in the supernatants corrected by the levels of FhaC produced by the recombinant strains. They were obtained by determining the ratios of the intensities of the Fha44 and FhaC bands on immunoblots, with the secretion activity of the strain producing WT FhaC set to 100%. The activities shown in the table correspond to a representative experiment. The mutant FhaCs were sorted in decreasing order of activities.b Conductances were determined in 1 m KCl using single channel recordings. NC indicates not calculated because signals were too noisy; ND indicates not determined; NA indicates no activity. Open table in a new tab We then sought to purify each of the mutant proteins in order to analyze their pore properties in lipid bilayers. To this end, WT FhaC as well as each of the mutant proteins were overproduced in a porin-deficient E. coli strain, BL21(DE3)-omp5, and the corresponding proteins were purified. Under those conditions, most mutant FhaCs were obtained at reasonable yields. However, FhaC-243B was obtained in very low amounts, whereas FhaC-33B could not be extracted from the membranes, most likely because it was significantly perturbed by the insertion. FhaC-33B was thus not considered further. Characterization of WT FhaC in Artificial Lipid Bilayers; Macroscopic I/V Curves—In a previous study, we reported that FhaC formed ion-permeable channels showing relatively brief openings and with conductance values around 1200 pS in 1 m KCl (21Jacob-Dubuisson F. El-Hamel C. Saint N. Guedin S. Willery E. Molle G. Locht C. J. Biol. Chem. 1999; 274: 37731-37735Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In the present study, we looked at ion channel properties of FhaC by recording macroscopic current-voltage (I/V) curves (Fig. 2A). In this configuration, up to a hundred channels are incorporated and submitted to slow voltage ramps at relatively high protein concentrations. Macroscopic I/V curves are useful to screen the functional properties of potential channel formers in lipid bilayers and allow relatively rapid comparisons between related proteins, i.e. mutants of FhaC. The I/V curves obtained after addition of FhaC at a final concentration around 100 ng/ml to the cis-side of azolectin bilayers submitted to slow triangular voltage ramps (10 mV/s) were asymmetric, with the apparition of a hysteresis in the negative quadrant (Fig. 2A). This result indicates that structurally asymmetric FhaC molecules, as suggested by secondary structure predictions, insert in a preferred orientation into lipid bilayers. However, the absolute orientation of the molecules could not be determined from these current-voltage recordings. The I/V curve in Fig. 2A exhibits a linear part from +100 mV to around –60 mV, indicating a voltage-independent conductance of FhaC channels in this voltage range, whereas a curve bending appeared from –60 to –100 mV in the increasingly negative voltage branch. This latter observation suggests the following. (i) The conductance characteristics of FhaC are dependent on the membrane potential above a threshold voltage (around –60 mV). (ii) A conformational change of FhaC channels, such as a displacement of loops, may appear at this particular voltage, which results in higher ion-conducting pores as observed from increasing currents recorded above this potential. It is noteworthy that the falling voltage branch in the negative part of the I/V curve did not present a curve bending as observed in the corresponding rising voltage branch. This indicates that the channels might not switch back to their initial conformation during the relaxation process at negative voltages. Characterization of Wild Type FhaC in Artificial Lipid Bilayers; Single-channel Measurements—In order to characterize further the FhaC channels and to confirm that the asymmetric properties in ion conduction observed from the I/V recordings were an intrinsic property of membrane-inserted FhaC, we also examined the influence of membrane potential on conductance in single-channel experiments. To this end, very small amounts of FhaC molecules were introduced into the cis-compartment bathing the lipid bilayer (final concentration around 0.1 to 1 ng/ml). Fig. 2B shows ionic currents through a single FhaC channel recorded at different membrane potentials. The current increased with increasing voltages between +40 and +80 mV, and the conductance value (about 1200 pS; see the amplitude histogram on Fig. 2C) remained stable, indicating that no voltage-dependent conductance occurred in this potential range. In contrast, the conductance characteristics of FhaC changed at negative voltages. Although altered conductance could not be well visualized at –40 mV, current recordings at –60 mV became slightly noisy and revealed that the FhaC channel occasionally displayed switches into substrates. This behavior became more pronounced at increasing negative potentials until the channel conductance appeared very noisy. Fig. 2D shows current recorded through an FhaC channel at repetitive pulses of +60 and –60 mV. The appearance of noisy currents during negative pulses is a consequence of applied negative voltages, because noisy channels did not persist when potentials were switched back to positive polarity. The increasing noise at high negative potentials could be because of an asymmetric distribution of positive and negative charges across the membrane combined with a conformational mobility of parts of the protein, as described earlier for TolC and engineered variants of LamB (30Andersen C. Bachmeyer C. Tauber H. Benz R. Wang J. Michel V. Newton S.M. Hofnung M. Charbit A. Mol. Microbiol. 1999; 32: 851-867Crossref PubMed Scopus (25) Google Scholar, 31Andersen C. Hughes C. Koronakis V. J. Membr. Biol. 2002; 185: 83-92Crossref PubMed Scopus (48) Google Scholar). The single-channel measurements thus confirm that the FhaC channels display asymmetric" @default.
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