FhuA deletion variant Δ1-159 overexpression in inclusion bodies and refolding with Polyethylene-Poly(ethylene glycol) diblock copolymer

Membrane protein isolation is a challenging problem. In fact especially their extraction from the respective membrane is difficult and often goes along with losses in yield. Usually expensive detergents are needed to extract the target protein from the membrane. Therefore finding an efficient overexpression and extraction method and an alternative to detergents is desirable. In this study we describe a new and fast method to express, extract and purify an engineered variant of the FhuA protein (FhuA Δ1-159) that acts as passive diffusion channel, using a diblock copolymer as an alternative to detergents like octyl-POE (n-octylpolyoxyethylene). The N-terminal leader sequence, facilitating the protein's transport to the outer membrane was deleted (FhuA Δ1-159 Δsignal), resulting in protein accumulation in easy to isolate inclusion bodies. Urea was used to solubilise the unfolded protein and dialysis against phosphate-buffer containing the commercially available diblock copolymer PE-PEG[Polyethylene-Poly(ethyleneglycol)] lead to protein refolding. Circular dichroism spectroscopy revealed a high β-sheet percentage within the refolded protein secondary structure indicating the successful reconstitution of FhuA Δ1-159 Δsignal native state. Furthermore the channel functionality of FhuA Δ1-159 Δsignal was verified by measuring the in and out-flux through the protein when inserted into liposome membrane, using the HRP/TMB (HRP=Horse Radish Peroxidase, TMB=3,3',5,5'-tetramethylbenzidine) assay system.     

Redesign of a plugged beta-barrel membrane protein

The redesign of biological nanopores is focused on bacterial outer membrane proteins and pore-forming toxins, because their robust β-barrel structure makes them the best choice for developing stochastic biosensing elements. Using membrane protein engineering and single-channel electrical recordings, we explored the ferric hydroxamate uptake component A (FhuA), a monomeric 22-stranded β-barrel protein from the outer membrane of Escherichia coli. FhuA has a luminal cross-section of 3.1 × 4.4 nm and is filled by a globular N-terminal cork domain. Various redesigned FhuA proteins were investigated, including single, double, and multiple deletions of the large extracellular loops and the cork domain. We identified four large extracellular loops that partially occlude the lumen when the cork domain is removed. The newly engineered protein, FhuAΔC/Δ4L, was the result of a removal of almost one-third of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. This extensive protein engineering encompassed the entire cork domain and four extracellular loops. Remarkably, FhuAΔC/Δ4L forms a functional open pore in planar lipid bilayers, with a measured unitary conductance of ～4.8 nanosiemens, which is much greater than the values recorded previously with other engineered FhuA protein channels. There are numerous advantages and prospects of using such an engineered outer membrane protein not only in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating, but also in more applicative areas of stochastic single-molecule sensing of proteins and nucleic acids.     

Insertion of bacteriorhodopsin into polymerized diacetylenic phosphatidylcholine bilayers

We have developed a method to incorporate the membrane protein bacteriorhodopsin into polymerized bilayers composed of a diacetylenic phosphatidylcholine, 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) and a non-polymerizable phospholipid, dinonanoylphosphatidylcholine (DNPC). The extent of DC8,9PC polymerization in the bilayer was significantly improved when 2:1 mole ratio DNPC-DC8,9PC was used. Octyl glucopyranoside-solubilized bacteriorhodopsin was inserted into the polymerized DNPC-DC8,9PC bilayers by overnight incubation at 4 degrees C followed by dialysis to remove the detergent. The protein was inserted into the membranes after photo-polymerization to avoid inactivation of the protein due to the UV irradiation. The insertion of bacteriorhodopsin into the polymerized DNPC-DC8,9PC membranes was confirmed by density gradient centrifugation, UV/visible spectroscopy, and freeze fracture electron microscopy. The polymerized DNPC-DC8,9PC membranes containing bacteriorhodopsin were about 10% protein by weight. These results suggest that mixed lipid systems such as the DNPC-DC8,9PC can be used to improve both the extent of polymerization and the efficiency of membrane protein incorporation in the polymerized bilayer.     

Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.

The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.     

Membrane protein dynamics versus environment: simulations of OmpA in a micelle and in a bilayer

The bacterial outer membrane protein OmpA is one of the few membrane proteins whose structure has been solved both by X-ray crystallography and by NMR. Crystals were obtained in the presence of detergent, and the NMR structure is of the protein in a detergent micelle. We have used 10 ns duration molecular dynamics simulations to compare the behaviour of OmpA in a detergent micelle and in a phospholipid bilayer. The dynamic fluctuations of the protein structure seem to be ca 1.5 times greater in the micelle environment than in the lipid bilayer. There are subtle differences between the nature of OmpA-detergent and OmpA-lipid interactions. As a consequence of the enhanced flexibility of the OmpA protein in the micellar environment, side-chain torsion angle changes are such as to lead to formation of a continuous pore through the centre of the OmpA molecule. This may explain the experimentally observed channel formation by OmpA.     

FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5.

The Escherichia coli outer membrane protein FhuA catalyzes the transport of Fe3+(-)ferrichrome and is the receptor of phage T5 and phi 80. The purified protein inserted into planar lipid bilayers showed no channel activity. Binding of phage T5 and FhuA resulted in the appearance of high conductance ion channels. The electrophysiological characteristics of the channels (conductance, kinetic behavior, substates, ion selectivity including the effect of ferrichrome) showed similarities with those of the channel formed by a FhuA derivative from which the 'gating loop' (delta 322-355) had been removed. binding of phage T5 to FhuA in E.coli cells conferred SDS sensitivity to the bacteria, suggesting that such channels also exist in vivo. These data suggest that binding of T5 to loop 322-355 of FhuA, which constitutes the T5 binding site, unmasks an inner channel in FhuA. Both T5 and ferrichrome bind to the closed state of the channel but only T5 can trigger its opening.     

Association of spin-labeled lipids with beta-barrel proteins from the outer membrane of Escherichia coli

The interaction of spin-labeled lipids with beta-barrel transmembrane proteins has been studied by the electron spin resonance (ESR) methods developed for alpha-helical integral proteins. The outer membrane protein OmpA and the ferrichrome-iron receptor FhuA from the outer membrane of Escherichia coli were reconstituted in bilayers of dimyristoylphosphatidylglycerol. The ESR spectra from phosphatidylglycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the beta-barrel, in addition to that from the fluid bilayer lipids. The stoichiometry of motionally restricted lipids, 11 and 32 lipids/monomer for OmpA and FhuA, respectively, is constant irrespective of the total lipid/protein ratio. It is proportional to the number of transmembrane beta-strands, eight for OmpA and 22 for FhuA, and correlates reasonably well with the intramembranous perimeter of the protein. Spin-labeled lipids with different polar headgroups display a differential selectivity of interaction with the two proteins. The more pronounced pattern of lipid selectivity for FhuA than for OmpA correlates with the preponderance of positively charged residues facing the lipids in the extensions of the beta-sheet and shorter interconnecting loops on the extracellular side of FhuA.     

FhuA barrel-cork hybrids are active transporters and receptors

The crystal structure of Escherichia coli FhuA reveals a beta-barrel domain that is closed by a globular cork domain. It has been assumed that the proton motive force of the cytoplasmic membrane through the interaction of the TonB protein with the TonB box of the cork opens the FhuA channel. Yet, deletion of the cork results in an FhuA derivative, FhuADelta5-160, that still displays TonB-dependent substrate transport and phage receptor activity. To investigate this unexpected finding further, we constructed FhuADelta5-160 derivatives of FhuA proteins from Salmonella paratyphi B, Salmonella enterica serovar Typhimurium, and Pantoea agglomerans. The FhuADelta5-160 proteins inserted correctly into the outer membrane, and with the exception of the P. agglomerans protein, transported ferrichrome and albomycin. FhuA hybrids consisting of the beta-barrel of one strain and the cork of another strain were active and showed higher TonB-dependent ferrichrome transport rates than the corkless derivatives. Exceptions were the E. coli beta-barrel/Salmonella serovar Typhimurium cork hybrid protein and the Salmonella serovar Typhimurium beta-barrel/P. agglomerans cork hybrid protein, both of which were less active than the beta-barrels alone. Each of the FhuA mutant proteins displayed activity for each of their ligands, except for phage T5, only when coupled to TonB. The hybrid FhuA proteins displayed a similar activity with the E. coli TonB protein as with their cognate TonB proteins. Sensitivity to phages T1, T5, and phi80, rifamycin CGP 4832, and colicin M was determined by the beta-barrel, whereas sensitivity to phage ES18 and microcin J25 required both the beta-barrel and cork domains. These results demonstrate that the beta-barrel domain of FhuA confers activity and specificity and responds to TonB and that the cork domains of various FhuA proteins can be interchanged and contribute to the activities of the FhuA hybrids.     

Voltage-induced gating of the mechanosensitive MscL ion channel reconstituted in a tethered lipid bilayer membrane

The mechanosensitive (MS) ion channel is gated by changes in bilayer deformation. It is functional without the presence of any other proteins and gating of the channel has been successfully achieved using conventional patch clamping techniques where a voltage has been applied together with a pressure over the membrane. Here, we have for the first time analyzed the large conducting (MscL) channel in a supported membrane using only an external electrical field. This was made possible using a newly developed technique utilizing a tethered lipid bilayer membrane (tBLM), which is part of an engineered microelectronic array chip. Single ion channel activity characteristic for MscL was obtained, albeit with lower conductivity. The ion channel was gated using solely a transmembrane potential of 300 mV. Computations demonstrate that this amount of membrane potential induces a membrane tension of 12 dyn/cm, equivalent to that calculated to gate the channel in patch clamp from pressure-induced stretching of the bilayer. These results strengthen the supposition that the MscL ion channel gates in response to stress in the lipid membrane rather than pressure across it. Furthermore, these findings illustrate the possibility of using the MscL as a release valve for engineered membrane devices; one step closer to mimicking the true function of the living cell.     

Inactivation in vitro of the Escherichia coli outer membrane protein FhuA by a phage T5-encoded lipoprotein

Bacteriophage T5-encoded lipoprotein, synthesized by infected Escherichia coli cells, prevents superinfection of the host cell by this virus. The molecular basis of its ability to inactivate the receptor of phage T5, the FhuA protein, was investigated in vitro. Fully competent T5 lipoprotein, with a His tag attached to the C-terminus, was purified in detergent solution. Co-reconstitution with homogeneous FhuA protein into liposomes revealed that the lipoprotein inhibited the irreversible inactivation of phage T5 by FhuA protein. This phenomenon correlated with the inhibition of phage DNA ejection determined by fluorescence monitoring. Addition of detergent abolished the interaction between T5 lipoprotein and FhuA protein. When the signal sequence and N-terminal cysteinyl residue of the lipoprotein were removed by genetic truncation, the soluble polypeptide could be refolded and purified from inclusion bodies. The truncated lipoprotein interfered with infection of E. coli by phage T5, but only at very high concentrations. Circular dichroism spectra of both forms of T5 lipoprotein exhibited predominantly β-structure. T5 lipoprotein is sufficient for inactivation of the FhuA protein, presumably by inserting the N-terminal acyl chains into the membrane, thus increasing its local concentration. An in vitro stoichiometry of 10:1 has been calculated for the phage-encoded T5 lipoprotein to FhuA protein complex.     

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.     

Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE

The preprotein translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two receptor components, Toc159 and Toc34, and the channel Toc75 form the Toc complex. In this study, we have analyzed the molecular architecture and organization of the Toc complex by blue native PAGE (BN-PAGE), which is a high-resolution method for separating membrane protein complexes under non-denaturing conditions. Pea chloroplasts isolated in the presence of a protease inhibitor cocktail were directly solubilized in detergent solution and analyzed by BN-PAGE and size exclusion chromatography. Subsequent immunoblot analyses indicated that the complex composed of Toc75, Toc159 and Toc34 has a molecular mass of 800-1,000 kDa. Limited proteolysis revealed a core of the Toc complex, which was resistant to proteases and detergent treatments. The stoichiometry of the three Toc proteins was calculated as approximately 1 : 3 : 3 between Toc159 : Toc75 : Toc34. We have also analyzed the Toc complex of etioplasts and root plastids. These plastids were found to have essentially the same sized Toc complex as that of the chloroplast.     

The β-barrel domain of FhuAΔ5-160 is sufficient for TonB-dependent FhuA activities of Escherichia coli

FhuA in the outer membrane of Escherichia coli serves as a transporter for ferrichrome, the antibiotics albomycin and rifamycin CGP4832, colicin M, and as receptor for phages T1, T5 and phi80. The previously determined crystal structure reveals that residues 160-714 of the mature protein form a beta-barrel that is closed from the periplasmic side by the globular N-proximal fragment, residues 1-159, designated the cork. In this study, deletion of the cork resulted in a stable protein, FhuADelta5-160, that was incorporated in the outer membrane. Cells that synthesized FhuADelta5-160 displayed a higher sensitivity to large antibiotics such as erythromycin, rifamycin, bacitracin and vancomycin, and grew on maltotetraose and maltopentaose in the absence of LamB. Higher concentrations of ferrichrome supported growth of a tonB mutant that synthesized FhuADelta5-160. These results demonstrate non-specific diffusion of compounds across the outer membrane of cells that synthesize FhuADelta5-160. However, growth of a FhuADelta5-160 tonB wild-type strain occurred at low ferrichrome concentrations, and ferrichrome was transported at about 45% of the FhuA wild-type rate despite the lack of ferrichrome binding sites provided by the cork. FhuADelta5-160 conferred sensitivity to the phages and colicin M at levels similar to that of wild-type FhuA, and to albomycin and rifamycin CGP 4832. The activity of FhuADelta5-160 depended on TonB, although the mutant lacks the TonB box (residues 7-11) previously implicated in the interaction of FhuA with TonB. CCCP inhibited tonB-dependent transport of ferrichrome through FhuADelta5-160. FhuADelta5-160 still functions as a specific transporter, and sites in addition to the TonB box are involved in the TonB-mediated response of FhuA to the proton gradient of the cytoplasmic membrane. It is proposed that TonB interacts with the TonB box of FhuA and with the beta-barrel to release ferrichrome from the FhuA binding sites and to open the channel in FhuA. For transport of ferrichrome through the open channel of FhuADelta5-160, interaction of TonB with the beta-barrel is sufficient to release ferrichrome from the residual binding sites at the beta-barrel and to induce the active conformation of the L4 loop at the cell surface for infection by the TonB-dependent phages T1 and phi80.     

Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins.

A considerable interest exists currently in designing innovative strategies to produce two-dimensional crystals of membrane proteins that are amenable to structural analysis by electron crystallography. We have developed a protocol for crystallizing membrane protein that is derived from the classical lipid-layer two-dimensional crystallization at the air/water interface used so far for soluble proteins. Lipid derivatized with a Ni(2+)-chelating head group provided a general approach to crystallizing histidine-tagged transmembrane proteins. The processes of protein binding and two-dimensional crystallization were analyzed by electron microscopy, using two prototypic membrane proteins: FhuA, a high-affinity receptor from the outer membrane of Escherichia coli, and the F(0)F(1)-ATP synthase from thermophilic Bacillus PS3. Conditions were found to avoid solubilization of the lipid layer by the detergent present with the purified membrane proteins and thus to allow binding of micellar proteins to the functionalized lipid head groups. After detergent removal using polystyrene beads, membrane sheets of several hundreds of square micrometers were reconstituted at the interface. High protein density in these membrane sheets allowed further formation of planar two-dimensional crystals. We believe that this strategy represents a new promising alternative to conventional dialysis methods for membrane protein 2D crystallization, with the additional advantage of necessitating little purified protein.     

An 8-A projected structure of FhuA, A "ligand-gated" channel of the Escherichia coli outer membrane.

The structure of FhuA, a siderophore and phage receptor in the outer membrane of Escherichia coli, has been investigated by electron crystallography. Bidimensional crystals of hexahistidine-tagged FhuA protein solubilized in N,N-dimethyldodecylamine-N-oxide were produced after detergent removal with polystyrene beads. Frozen-hydrated crystals (unit cell dimensions of a = 124 A, b = 98 A, gamma = 90 degrees ) exhibited a p22121 plane group symmetry. A projection map at 8 A resolution showed the presence of dimeric ring-like structures with an elliptical shape (48 x 40 A). Each monomer was composed of a ring of densities with a radial width of 8-10 A corresponding to a cylinder of beta sheets. Few densities are present inside the barrel, leaving a central channel approximately 25 A in diameter. A projection map of FhuA at 15 A resolution, which was calculated from negatively stained preparations, demonstrated that most of the central channel was masked by extramembrane domains. This map also revealed an asymmetric distribution of extramembrane domains in FhuA, with large domains located mainly on one side of the molecule. Comparison with density maps derived from recent atomic structure allowed further interpretation of the electron microscopy projection structures with regard to long hydrophilic loops governing the selectivity and opening of the channel.     

Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop.

Escherichia coli transports Fe3+ as a ferrichrome complex through the outer membrane in an energy-dependent process mediated by the FhuA protein. A FhuA deletion derivative lacking residues 322 to 355 (FhuA delta322-355) forms a permanently open channel through which ferrichrome diffused. This finding led to the concept that the FhuA protein forms a closed channel that is opened by input of energy derived from the electrochemical potential across the cytoplasmic membrane, mediated by the Ton system. In this study, we constructed various FhuA derivatives containing deletions inside and outside the gating loop. FhuA delta322-336 bound ferrichrome and displayed a residual Ton-dependent ferrichrome transport activity. FhuA delta335-355 no longer bound ferrichrome but supported ferrichrome diffusion through the outer membrane in the absence of the Ton system. FhuA delta335-355 rendered cells sensitive to sodium dodecyl sulfate and supported diffusion of maltotetraose and maltopentaose in a lamB mutant lacking the maltodextrin-specific channel in the outer membrane. Cells expressing FhuA delta70-223, which has a large deletion outside the gating loop, were highly sensitive to sodium dodecyl sulfate and grew on maltodextrins but showed only weak ferrichrome uptake, suggesting formation of a nonspecific pore through the outer membrane. FhuA delta457-479 supported Ton-dependent uptake of ferrichrome. None of these FhuA deletion derivatives formed pores in black lipid membranes with a stable single-channel conductance. Rather, the conductance displayed a high degree of current noise, indicating a substantial influence of the deletions on the conformation of the FhuA protein. FhuA also supports infection by the phages T1, T5, and phi80 and renders cells sensitive to albomycin and colicin M. Cells expressing FhuA delta322-336 were sensitive to albomycin and colicin M but were only weakly sensitive to T5 and phi480 and insensitive to T1. Cells expressing FhuA delta335-355 were resistant to all FhuA ligands. These results indicate different structural requirements within the gating loop for the various FhuA ligands. Cells expressing FhuA delta457-479 displayed a strongly reduced sensitivity to all FhuA ligands, while cells expressing FhuA delta70-223 were rather sensitive to all FhuA ligands except albomycin, to which they were nearly resistant. It is concluded that residues 335 to 355 mainly determine the properties of the gate with regard to FhuA permeability and ligand binding.     

MD simulations of Mistic: conformational stability in detergent micelles and water

Mistic is an unusual membrane protein from Bacillus subtilis. It appears to fold and insert autonomously into a lipid bilayer and has been suggested as a tool that aids the targeting of eukaryotic membrane proteins to bacterial membranes. The NMR structure of Mistic in detergent (LDAO) micelles has revealed it to be a four alpha-helix bundle. From a structural perspective, Mistic does not resemble other membrane proteins. Its external surface is not very hydrophobic, and standard methods do not predict any of its helices to be in the transmembrane orientation. Molecular dynamics simulations (simulation times approximately 30 ns) in water and in detergent micelles have been used to explore the conformational stability of Mistic as a function of its environment. In water, the protein is stable, exhibiting no significant change in fold on a 30 ns time scale. In contrast, in three simulations in detergent micelles, the partial unfolding of Mistic occurred, whereby the H4 helix drifted away from the H1-H3 core. This was due to the penetration of detergent molecules between H4 and the remainder of the protein. This is unlike the behavior of several other membrane proteins, both alpha-helix bundles and beta-barrels, in comparable detergent micelle simulations. The unfolding of H4 from the H1-H3 core of Mistic could be partially reversed by a simulation in which the detergent molecules were removed, and the unfolded protein was simulated in water. These results suggest that Mistic may not be a stable integrated membrane protein but rather that it may undergo a conformational change upon interaction with a membrane or membrane-like environment.     

Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12.

The ferrichrome-iron receptor of Escherichia coli K-12 is FhuA (M(r), 78,992), the first component of an energy-dependent, high-affinity iron uptake pathway. FhuA is also the cognate receptor for bacteriophages T5, T1, phi 80, and UC-1, for colicin M and microcin 25, and for albomycin. To probe the topological organization of FhuA which enables recognition of these different ligands, we generated a library of 16 insertion mutations within the fhuA gene. Each insertion spliced a 13-amino-acid antigenic determinant (the C3 epitope of poliovirus) at a different position within FhuA. Immunoblotting of outer membranes with anti-FhuA and anti-C3 antibodies indicated that 15 of 16 FhuA.C3 proteins were present in the outer membrane in amounts similar to that observed for plasmid-encoded wild-type FhuA. One chimeric protein with the C3 epitope inserted after amino acid 440 of FhuA was present in the outer membrane in greatly reduced amounts. Strains overexpressing FhuA.C3 proteins were subjected to flow cytometric analysis using anti-FhuA monoclonal antibodies. Such analysis showed that (i) the chimeric proteins were properly localized and (ii) the wild-type FhuA protein structure had not been grossly altered by insertion of the C3 epitope. Twelve of sixteen strains expressing FhuA.C3 proteins were proficient in ferrichrome transport and remained sensitive to FhuA-specific phages. Three FhuA.C3 proteins, with insertions after amino acid 321, 405, or 417 of FhuA, were detected at the cell surface by flow cytometry using anti-C3 antibodies. These three chimeric proteins were all biologically active. We conclude that amino acids 321, 405, and 417 are surface accessible in wild-type FhuA.     

CLIC1 inserts from the aqueous phase into phospholipid membranes, where it functions as an anion channel

CLIC1 is a member of the CLIC familyof proteins, which has been shown to demonstrate chloride channelactivity when reconstituted in phospholipid vesicles. CLIC1 exists incells as an integral membrane protein and as a soluble cytoplasmicprotein, implying that CLIC1 might cycle between membrane-inserted andsoluble forms. CLIC1 was purified and detergent was removed, yieldingan aqueous solution of essentially pure protein. Pure CLIC1 was mixedwith vesicles, and chloride permeability was assessed with a chloride efflux assay and with planar lipid bilayer techniques. Soluble CLIC1confers anion channel activity to preformed membranes that isindistinguishable from the previously reported activity resulting fromreconstitution of CLIC1 into membranes by detergent dialysis. Theactivity is dependent on the amount of CLIC1 added, appears rapidly onmixing of protein and lipid, is inhibited by indanyloxyacetic acid-94,N-ethylmaleimide, and glutathione, is inactivated by heat,and shows sensitivity to pH and to membrane lipid composition. Weconclude that CLIC1 in the absence of detergent spontaneously insertsinto preformed membranes, where it can function as an anion-selective channel.