Interaction of bacteriophage lambda with its cell surface receptor: an in vitro study of binding of the viral tail protein gpJ to LamB (Maltoporin)

The cell surface receptor for bacteriophage Lambda is LamB (maltoporin). Responsible for phage binding to LamB is the C-terminal part, gpJ, of phage tail protein J. To study the interaction between LamB and gpJ, a chimera protein composed of maltose binding protein (MBP or MalE) connected to the C-terminal part of J (gpJ, amino acids 684-1131) of phage tail protein J of bacteriophage Lambda was expressed in Escherichia coli and purified to homogeneity. The interaction of the MBP-gpJ chimera protein with reconstituted LamB and its mutants LamB Y118G and the loop deletion mutant LamB Delta4+Delta6+Delta9v was studied using planar lipid bilayer membranes on a single-channel and multichannel level. Titration with the MBP-gpJ chimera blocked completely the ion current through reconstituted LamB when it was added to the cis side, the extracellular side of LamB with a half-saturation constant of approximately 6 nM in 1 M KCl. Control experiments with LamB Delta4+Delta6+Delta9v from which all major external loops had been removed showed similar blocking, whereas MBP alone caused no visible effect. Direct conductance measurement with His(6)-gpJ that contained a hexahistidyl tag (His(6) tag) at the N-terminal end of the protein for easy purification revealed no blocking of the ion current, requiring other measurements for the binding constant. However, when maltoporin was preincubated with His-gpJ, MBP-gpJ could not block the channel, which indicated that also His(6)-gpJ bound to the channel. High-molecular mass bands on SDS-PAGE and Western blots, confirming the planar lipid bilayer experiment results, also demonstrated stable complex formation between His(6)-gpJ and LamB or LamB mutants. The results revealed that phage Lambda binding includes not only the extracellular loops.     

Probing the orientation of reconstituted maltoporin channels at the single-protein level

Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding. This asymmetry revealed also that maltoporin insertion is directional. However, the orientation in the lipid bilayer remained an open question. To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition. In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side. In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition. Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions. We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side.     

In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K-12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding

The trimeric protein LamB of Escherichia coli K-12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18-stranded antiparallel beta-barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro. In vivo, single-, DeltaL4, DeltaL5, DeltaL6, and double-loop deletions, DeltaL4 + DeltaL5 and DeltaL5 + DeltaL6, abolished maltoporin functions, but not the double deletion DeltaL4 + DeltaL6 and the triple deletion DeltaL4 + DeltaL5 + DeltaL6. While deletion of the central variable portion of loop L9 (DeltaL9v) affected maltoporin function only moderately, the combination of DeltaL9v with the double deletion of loops L4 and L6 (triple deletion DeltaL4 + DeltaL6 + DeltaL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate-binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBDeltaL9v), of two loops L4 and L6 (LamBDeltaL4 + DeltaL6), of three loops L4, L5 and L6 (LamBDeltaL4 + DeltaL5 + DeltaL6) or of L4, L6 and L9v (LamBDeltaL4 + DeltaL6 + DeltaL9v) had relatively little influence on the carbohydrate-binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBDeltaL4) or L6 (LamBDeltaL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the purification across the starch column, suggest that maltooligosaccharides enter the LamB channel from the cell surface side with the non-reducing end in advance. The absence of some of the loops leads to obstruction of the channel from the outside, which results in a considerable difference in the on-rate of carbohydrate binding from the extracellular side compared with that from the periplasmic side.     

High-conductance channel induced by the interaction of phage lambda with its receptor maltoporin

Bacteriophage lambda that binds to liposomes bears its receptor maltoporin (LamB) and is able to inject its DNA into the internal space. During this process, the liposomes are permeabilized, suggesting that a transmembrane channel has formed (Roessner and Ihler (1986) J. Biol. Chem. 261, 386-390). This pore possibly constitutes the pathway used by lambda DNA to cross the membrane. We reconstituted purified LamB from Shigella in liposomes that were incubated with lambda phages. Addition of this mixture to a bilayer chamber resulted in the incorporation in planar bilayers of high-conductance channels whose conductance, kinetics and voltage dependence were totally different from those of maltoporin channels.     

Antibodies affect the ionic conductance of channels formed by amphotericin B in a lipid bilayer

For the first time poly- and monoclonal antibodies (class IgM) against the polyene antibiotic amphotericin B were obtained affecting the properties of a channel formed by the antibiotic and cholesterol in a lipid bilayer when amphotericin B was added to the solution at one (cis) side of the membrane. In the case of the symmetric distribution of cholesterol in the lipid bilayer, three molecules of monoclonal antibodies bind firmly to the channel at the trans-side of the membrane, thus strongly increasing the mean lifetime of the channel in the open state, and not changing practically the ion conductance of its open state. The antibodies did not alter the properties of these channels when added at the cis-side of the membrane as well as of the channels formed in the lipid bilayer when amphotericin B was added at both membrane sides. The antibodies obtained did not affect the conductance of channels in which amphotericin B and cholesterol were replaced with their analogs levorin and 5 alpha-androstan-3 beta-one, which points to a high specificity of the immunoglobulins isolated. When cholesterol was present only in the cis-monolayer of the lipid bilayer and was absent in the trans-monolayer, the same monoclonal antibodies when added at the trans-side of the membrane blocked the conductance of the channel formed by adding the antibiotic to the solution at the cis-side of the bilayer. The obtained evidence is of interest in elucidating the general features of interaction of antibodies with the ionic channels of cellular and model membranes.     

LamB (maltoporin) of Salmonella typhimurium: isolation, purification and comparison of sugar binding with LamB of Escherichia coli

LamB (maltoporin) of Salmonella typhimurium was found to be more strongly associated with the murein than OmpF. It was purified in one step using a hydroxyapatite (HTP) column. Reconstitution of the pure protein with lipid bilayer membrane showed that LamB of S. typhimurium formed small ion-permeable channels with a single channel conductance of about 90 pS in 1 M KCl and some preference for cations over anions. The conductance concentration curve was linear, which suggested that LamB of S. typhimurium does not contain any binding site for ions. Pore conductance was completely inhibited by the addition of 20 mM maltotriose. Titration of the LamB-induced membrane conductance with different sugars, including all members of the maltooligosaccharide series up to seven glucose residues, suggested that the channel contains, like LamB (maltoporin) of Escherichia coli, a binding site for sugars. The binding constant of sugars of the maltooligosaccharide series increased with increasing number of glucose residues up to five (saturated). Small sugars had a higher stability constant for sugar binding relative to LamB of E. coli. The advantage of a binding site inside a specific porin for the permeation of solutes is discussed with respect to the properties of a general diffusion porin.     

Oriented channels reveal asymmetric energy barriers for sugar translocation through maltoporin of Escherichia coli.

Sugar transport through maltoporin of Escherichia coli was investigated. This protein facilitates maltooligosaccharide translocation via a binding site in the channel. Because incorporation of the protein into the bilayer results in randomly orientated channels, we re-examined the postulated symmetric translocation model by reconstitution of maltoporin under an externally applied field. Upon binding of bacteriophage lambda, which exploit surface-exposed loops of maltoporin as the receptor, sugar permeation, but not the ion current, was blocked. Thus using the phage-to-probe orientation we were able to show that the channels were approximately 80% directionally inserted into the bilayer. Moreover, asymmetry of the channel was revealed because sugar entrance through the 'open' periplasmic side of maltoporin was similarly reduced. Here a new asymmetrical two-barrier model is presented. Based on liposome-swelling assays and current-fluctuation analysis we conclude that the periplasmic side of the porin shows a two- to threefold higher energy barrier than the extracellular loop-side of the channels.     

Vitamin B1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes

The vitamin B1 (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(beta-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl2 and KCl solutions via ionic channels produced by latrotoxins (alpha-latrotoxin (alpha-LT) and alpha-latroinsectotoxin (alpha-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl2 inhibited transmembrane current by 31.6+/-3% and by 61.8+/-3% from the trans-side of BLM for alpha-LT channels. Application of DMHT in the solution of 10 mM CaCl2 to the cis-side of BLM decreased the current through the alpha-LIT and RTX channels by 52+/-4% and 50+/-5%, respectively. Addition of Cd2+ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca2+-current across the alpha-LT channels caused its further decrease by 85+/-5% that coincides favorably with the intensity of Cd2+ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on alpha-LT channels without considerable influence on the ionogenic groups of Ca2+-selective site inside the channel cavity. The binding kinetics of DMHT with the alpha-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of alpha-LT channel on either side of the BLM. The comparative analysis of effective radii measured for alpha-LT, alpha-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for alpha-LT channel on the trans-side (0.28+/-0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.     

A model of maltodextrin transport through the sugar-specific porin, LamB, based on deletion analysis.

LamB facilitates the uptake of maltose and maltodextrins across the bacterial outer membrane and acts as a general porin for small molecules. Using directed deletion mutagenesis we removed several regions of the LamB polypeptide and identified a polypeptide loop that both constricts the maltoporin channel and binds maltodextrins. In conjunction with a second sugar binding site that we identified at the rim of the channel, these data clarify, for the first time, the mechanism of transport through a substrate-specific porin. Furthermore, unlike the transverse loops of general porins, which originate from a central location in their primary structure, the loop that regulates LamB permeability originates from a C-terminal site. Thus LamB represents a second distinct class of porins in the bacterial outer membrane that is differently organized and separately evolved from OmpF-type, general porins.     

Evaluation of the rate constants of sugar transport through maltoporin (LamB) of Escherichia coli from the sugar-induced current noise

LamB (maltoporin) of Escherichia coli outer membrane was reconstituted into artificial lipid bilayer membranes. The channel contains a binding site for sugars and is blocked for ions when the site is occupied by a sugar. The on and off reactions of sugar binding cause an increase of the noise of the current through the channel. The sugar-induced current noise of maltoporin was used for the evaluation of the sugar-binding kinetics for different sugars of the maltooligosaccharide series and for sucrose. The on rate constant for sugar binding was between 10(6) and 10(7) M-1.s-1 for the maltooligosaccharides and corresponds to the movement of the sugars from the aqueous phase to the central binding site. The off rate (corresponding to the release of the sugars from the channel) decreased with increasing number of glucose residues in the maltooligosaccharides from approximately 2,000 s-1 for maltotriose to 180 s-1 for maltoheptaose. The kinetics for sucrose movement was considerably slower. The activation energies of the stability constant and of the rate constants for sugar binding were evaluated from noise experiments at different temperatures. The role of LamB in the transport of maltooligosaccharides across the outer membrane is discussed.     

Site-directed mutagenesis of tyrosine 118 within the central constriction site of the LamB (maltoporin) channel of Escherichia coli. II. Effect on maltose and maltooligosaccharide binding kinetics

The 3-D structure of the maltooligosaccharide-specific LamB channel of Escherichia coli (also called maltoporin) is known from x-ray crystallography. The central constriction of the channel formed by the external loop 3 is controlled by tyrosine 118. Y118 was replaced by site-directed mutagenesis by 10 other amino acids (alanine (A), isoleucine (I), asparagine (N), serine (S), cysteine (C), aspartic acid (D), arginine (R), histidine (H), phenylalanine (F), and tryptophan (W)) including neutral ones, negatively and positively charged amino acids to study the effect of their size, their hydrophobicity index, and their charge on maltose and maltooligosaccharide binding to LamB. The mutants were reconstituted into lipid bilayer membranes and the stability constants for binding of maltose, maltotriose, maltopentaose, and maltoheptaose to the channel were measured using titration experiments. The mutation of Y118 to any other non-aromatic amino acid led to a substantial decrease of the stability constant of binding by factors between about two and six. The highest effect was observed for the mutant Y118A. Replacement of Y118 by the two other aromatic amino acids, phenylalanine (F) and tryptophan (W), resulted in a substantial increase of the stability constant maximally by a factor of almost 400 for the Y118W mutant. The carbohydrate-induced block of the channel function was used for the study of current noise through the different mutant LamB channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k(1) and k(-1)) of sugar binding. The results suggest that both rate constants were affected by the mutations. For most mutants, with the exception of Y118F and Y118W, k(1) decreased and k(-1) increased, whereas the opposite was found for the aromatic amino acid mutants. The results suggest that tyrosine 118 has a crucial effect on carbohydrate transport through LamB.     

The sugar-specific outer membrane channel ScrY contains functional characteristics of general diffusion pores and substrate-specific porins

Escherichia coli K-12 strain PS1-28-37 carries the multicopy plasmid pPSO28-37 containing a DNA fragment coding for two of the proteins that enable bacteria to utilize sucrose as sole carbon source. One of the different gene products of the plasmid is the outer membrane protein, ScrY. This protein was isolated and purified by chromatography across a gel filtration column. Reconstitution experiments with lipid bilayer membrane demonstrated that ScrY formed ion-permeable channels with properties very similar to those of general diffusion pores of enteric bacteria. The presence of sugars in the aqueous phase led to a dose-dependent block of ion transport through the channel, like the situation found with LamB (maltoporin) of Escherichia coli and Salmonella typhimurium. The binding constants of a variety of different sugars were determined. The stability constant for malto-oligosaccharide binding increased with increasing numbers of glucose residues. Disaccharides generally had a larger binding constant than monosaccharides. The binding of different sugars to ScrY and LamB of E. coli is discussed with respect to the kinetics of sugar movement through the channel.     

Vitamin B1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes

The vitamin B₁ (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(β-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl₂ and KCl solutions via ionic channels produced by latrotoxins (α-latrotoxin (α-LT) and α-latroinsectotoxin (α-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl₂ inhibited transmembrane current by 31.6 ± 3% and by 61.8 ± 3% from the trans-side of BLM for α-LT channels. Application of DMHT in the solution of 10 mM CaCl₂ to the cis-side of BLM decreased the current through the α-LIT and RTX channels by 52 ± 4% and 50 ± 5%, respectively. Addition of Cd²⁺ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca²⁺-current across the α-LT channels caused its further decrease by 85 ± 5% that coincides favorably with the intensity of Cd²⁺ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on α-LT channels without considerable influence on the ionogenic groups of Ca²⁺-selective site inside the channel cavity. The binding kinetics of DMHT with the α-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of α-LT channel on either side of the BLM. The comparative analysis of effective radii measured for α-LT, α-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for α-LT channel on the trans-side (0.28 ± 0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.     

Mechanism of sugar transport through the sugar-specific LamB channel ofEscherichia coli outer membrane

Summary Lipid bilayer experiments were performed with the sugar-specific LamB (maltoporin) channel ofEscherichia coli outer membrane. Single-channel analysis of the conductance steps caused by LamB showed that there was a linear relationship between the salt concentration in the aqueous phase and the channel conductance, indicating only small or no binding between the ions and the channel interior. The total or the partial blockage of the ion movement through the LamB channel was not dependent on the ion concentration in the aqueous phase. Both results allowed the investigation of the sugar binding in more detail, and the stability constants of the binding of a large variety of sugars to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the sugars. The channel was highly cation selective, both in the presence and absence of sugars, which may be explained by the existence of carbonyl groups inside the channel. These carbonyl groups may also be involved in the sugar binding via hydrogen bonds. The kinetics of the sugar transport through the LamB channel were estimated relative to maltose by assuming a simple one-site, two-barrier model from the relative rates of permeation taken from M. Luckey and H. Nikaido (Proc. Natl. Acad. Sci. USA 77:165–171 (1980a)) and the stability constants for the sugar binding given in this study.     

Effect of point mutations on the in-vitro pore properties of maltoporin, a protein of Escherichia coli outer membrane

Maltoporin (LamB protein), a protein of Escherichia coli outer membrane forms ionic channels with a selectivity for maltose and maltodextrins (Dargent et al., 1987). The effect of different point mutations on maltoporin pore properties was investigated in vitro with planar bilayers. The mutations belong to three classes in terms of selective maltose transport in vivo: class A (substitution at positions 259 and 382) does not affect maltose transport, class B (position 163 and 245) decreases maltose transport down to 20 to 30%, and class C (position 18) almost completely abolishes selective maltose transport. This in-vitro study reveals that class A does not affect the pore properties in contrast to class B substitutions. The class B maltoporins are still able to form channels but display some specific features and altered specificity for maltose and maltodextrins. The substitution (Gly18----Val) alters trimer stability and impedes pore function (class C mutant). Thus, there is a good correlation between the specific transport properties of the mutated maltoporins in vivo and their behavior in vitro. These data, in combination with the asymmetric orientation of the protein within the bilayer and topological considerations, indicate that residues 245 and 163 do not belong to the selectivity filter. Mutations at these sites cause hindrance at the mouth of the pore on the outer domain of maltoporin.     

Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior.

LamB, a sugar-specific channel of Escherichia coli outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated using fast Fourier transformation. The current noise through the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz. The spectral density of the noise of the open LamB channels was a quadratic function of the applied voltage. Its magnitude was not correlated to the number of channels in the lipid bilayer membrane. Upon addition of sugars to the aqueous phase the current decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the sugars with the binding site inside the channel. The frequency dependence of the spectral density was of Lorentzian type, although the power of its frequency dependence was not identical to -2. Analysis of the power density spectra using a previously proposed simple model (Benz, R., A. Schmid, and G. H. Vos-Scheperkeuter. 1987. J. Membr. Biol. 100: 12-29), allowed the evaluation of the on- and the off-rate constants for the maltopentaose binding to the binding site inside the LamB channels. This means also that the maltopentaose flux through the LamB channel could be estimated by assuming a simple one-site, two-barrier model for the sugar transport from the results of the noise analysis.     

The gene bglH present in the bgl operon of Escherichia coli, responsible for uptake and fermentation of beta-glucosides encodes for a carbohydrate-specific outer membrane porin

The cryptic gene bglH from the Escherichia coli chromosome was cloned into a tacOP-driven expression vector. The resulting plasmid was transferred into the porin-deficient E. coli strain KS26 and the protein was expressed by addition of IPTG. The BglH protein was localized in the outer membrane. It was purified to homogeneity using standard methods. Reconstitution experiments with lipid bilayer membranes defined BglH as a channel-forming component, i.e. it is an outer membrane porin. The single-channel conductance of BglH (560 pS in 1 M KCl) was only one-third of that of the general diffusion porins of E. coli outer membrane. The presence of carbohydrates in the aqueous phase led to a dose-dependent block of ion transport through the channel, similar to that found for LamB (maltoporin) of E. coli and Salmonella typhimurium, which means that BglH is a porin specific for the uptake of carbohydrates. The binding constants of a variety of different carbohydrates were calculated from titration experiments of the BglH-induced membrane conductance. The tightest binding was observed with the aromatic beta-D-glucosides arbutin and salicin, and with gentibiose and cellobiose. Binding of maltooligosaccharides to BglH was in contrast to their binding to LamB in that it was much weaker, indicating that the binding site of BglH for carbohydrates is different from that of LamB (maltoporin). The kinetics of cellopentaose binding to BglH was investigated using the carbohydrate-induced current noise and was compared with that of cellopentaose binding to LamB (maltoporin) and ScrY (sucroseporin).     

N-terminal insertion of alamethicin in channel formation studied using its covalent dimer N-terminally linked by disulfide bond

Alamethicin is supposed to form helix-bundle-type channels by inserting the N terminus into bilayer lipid membranes under sufficient voltages. The N-terminal insertion has been studied with an alamethicin dimer (di-alm) N-terminally linked by a disulfide bond and by the asymmetric addition of dithiothreitol (DTT) and tetrathionate (TT) to the membrane. When di-alm was added to the cis-side membrane, it forms long-lasting channels with the lifetime tau of about 100 ms at cis-positive voltages. The lifetime was reduced to a few milliseconds by addition of DTT to the cis-side membrane, indicating that most of the channels were formed by the monomers (alm-SH) that resulted from the cleavage of the disulfide bond in di-alm. The succeeding addition of TT to the trans-side produced channels of tau=10-20 ms besides the channels of alm-SH. The results suggested that TT reacted with the N-terminal thiol group of alm-SH located at the trans-side of the membrane to alter the lifetime. The N-terminal insertion of alamethicin helices by voltage activation, therefore, was confirmed.     

In vivo and in vitro studies of transmembrane beta-strand deletion, insertion or substitution mutants of the Escherichia coli K-12 maltoporin

LamB of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane. Each monomer is composed of an 18-stranded antiparallel beta-barrel. In the present work, on the basis of the known X-ray structure of LamB, the effects of modifications of the beta-barrel domain of maltoporin were studied in vivo and in vitro. We show that: (i) the substitution of the pair of strands beta13-beta14 of the E. coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of beta-strands (deletion beta13-beta14) from the E. coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E. coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro. We also inserted into deletion beta13-beta14 the portion of the E. coli LamB protein comprising strands beta13 to beta16. This resulted in a protein expected to have 20 beta-strands and which completely lost all LamB-specific activities in vivo and in vitro.     

The thiazole derivative reduces transmembrane currents via ionic channels formed by alpha-latrotoxin and sea anemone toxin in the bilayer lipid membranes

It was shown that the thiazole derivative 3-decyloxycarbonylmethyl-4-methyl- 5-(2-hydroxyethyl)thiazole chloride (DMHT) (0.1 mM) reversibly reduced the transmembrane current in solutions of 10 mM CaCl2 and 100 mM KCl via ionic channels produced by alpha-latrotoxin from black widow spider (alpha-LT) and sea anemone toxin (RTX) in the bilayer lipid membranes (BLM). Introduction of DMHT from the cis-side of BLM inhibited transmembrane current by 31.6 +/- 3% and by 61.8 +/- 3% from the trans-side of BLM for alpha-LT channels. Application of DMHT to the cis-side BLM decreased the inward current through the RTX channels by 50 +/- 5%. Addition of Cd(2+) (0.1 mM) to the cis- or trans-side of a membrane after the DMHT induced depression of transmembrane current across the alpha-LT channels caused its further decrease by 85 +/- 5% that coincides completely with the intensity of Cd(2+)-inhibition in the control experiments without DMHT. These data suggest that DMHT may exert its inhibitory action on alpha-LT channels without considerable influence on the ionogenic groups inside the channel cavity. The comparative analysis of effective radii measured for alpha-LT and RTX channels on the cis- (0.9 nm and 0.55 nm, respectively) and the trans-side of BLM (< 0.467 nm for alpha-LT) allowed to propose the blocking action of DMHT for alpha-LT and RTX channels to result from direct penetration into the channel, achieved due to similar hydrodynamic size of blocking molecules and the size of toxin pores.