Moraxella catarrhalis is a gram-negative respiratory pathogen that is an important causative agen... more Moraxella catarrhalis is a gram-negative respiratory pathogen that is an important causative agent for otitis media and exacerbations of chronic obstructive pulmonary disease. We have previously predicted the outer membrane protein M35 to be a general porin, and in the current study, we have investigated the function of M35 and its importance for survival of M. catarrhalis in vivo. Lipid bilayer experiments reveal that refolded M35 functions as a channel that is typical of gram-negative bacterial porins. M35 forms wide and water-filled channels with a single-channel conductance of about 1.25 nS in 1 M KCl solution and has only a small selectivity for cations over anions. When the in vitro growth characteristics of two M35 deletion mutant strains of M. catarrhalis were compared to the wild-type parent isolates, the growth of the mutant strains was inhibited only under nutrient-poor conditions. This growth defect could be eliminated by additional glutamic acid, but not additional aspartic acid, glycine, sucrose, or glucose. The mutant strains compensated for the lack of M35 by enhancing their uptake of glutamic acid, and this enhanced rate of glutamic acid uptake was attributed to the compensatory upregulation of a protein of approximately 40 kDa. M35 was also found to be essential for nasal colonization of mice, demonstrating that its presence is essential for survival of M. catarrhalis in vivo. These results suggest that M35 is a general porin that is necessary for the uptake of important energy sources by M. catarrhalis and that it is likely that M35 is an essential functional protein for in vivo colonization.
Publisher Summary This chapter describes the relaxation techniques and noise analysis of ion carr... more Publisher Summary This chapter describes the relaxation techniques and noise analysis of ion carriers in planar bilayers. Only limited information on the carrier system can be obtained from stationary conductance measurements. Relaxation techniques may be used for the evaluation of the rate constants of the single transport steps. In a relaxation experiment an external parameter, such as pressure, temperature, or voltage, is quickly changed; after the perturbation the approach of the system toward a new stationary state is followed. In the analysis of relaxation experiments with alkali ion carders, it is usually assumed that the association and dissociation reactions in the interface are voltage independent and that the only transport step affected by the electric field in the membrane is the translocation of MS + . Analysis of current noise represents an alternative method for evaluating the rate constants of a carrier system. A straightforward method of extracting information from the fluctuating part of the current δI ( t ) consists in measuring the spectral intensity of current noise, because a function of frequency is the value of δI ( t ) 2 per unit frequency interval.
The major outer membrane protein of Rhodobacter capsulatus 37 b4 (capsule-free) was isolated. St... more The major outer membrane protein of Rhodobacter capsulatus 37 b4 (capsule-free) was isolated. Strong porin-activity was observed after reconstitution into artificial lipid bilayer membranes with a single channel conductance of 3.15 nS in Im KC1. The porin migrated as a broad, single band (Mr above 90,000) on sodium dodecyl sulfate polyacrylamide gel electro phoresis and dissociated into a single species of polypeptides (Mr 36,000) on treatment with EDTA (10 mM at 30 °C, 20 min) or by heating (100 °C, 5 min). Analytical ultracentrifugation studies demonstrated the native porin to be a trimer. The monomers chromatofocused as a single, sharp peak on fast performance liquid chromatography and only one band, corre sponding to an isoelectric point of about 4.0, was obtained on isoelectric focusing. Gas-phase sequencing of the 23 N-terminal residues revealed Glu-Val-Lys-Leu-Ser-Gly-Asp-Ala-Arg-Met-Gly-Val-Met-Tyr-Asn-Gly-Asp-Asp-X-Asn-Phe-Ser-Ser.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988
The mechanism of reversible electric breakdown of lipid membranes is studied. The following stage... more The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0,6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.
The sucrose-specific outer membrane parin ScrY of Salmonella typhimurium was isolated from EscMri... more The sucrose-specific outer membrane parin ScrY of Salmonella typhimurium was isolated from EscMrichia coti K-12 strain KS 26 containing the plasmid pPSOI12. The protein was purified to homogeneity by differential extraction of the cell envelope in the presence of the detergents sodium dodecyl sulfate and lauryl (dimethyl)-amine oxide (LDAO). The porin had apparent molecular weights of 58 kDa and 120 kDa for the monomer and for the trimer, respectively, on SDS/PAGE. The purified trimers were crystallized using poly(ethylene glycol) 2000 and the detergents octylglucoside (OG) and hexyl-(dimethyl)-amine oxide (C6DAO). X-ray diffraction of the crystals showed reflections to 2'3 A. The space group of the crystals was R3 and the lattice constants of the hexagonal axes were a = b = 112'85 A and c = 149'9 A. The crystal volume per unit of protein molecular weight was 3'47 A'jDa.
There is now considerable evidence that the ion permeability of biological membranes is not a dif... more There is now considerable evidence that the ion permeability of biological membranes is not a diffuse property of the whole membrane but depends on the presence of specialized components in the membrane. Such a functional component may be a mobile carrier molecule that mediates the transport of ions in a sequence of complexation, translocation, and decomplexation steps. The second limiting case of an ion transport mechanism consists of a porelike channel that offers to the ion a hydrophilic pathway through the apolar core of the membrane. The formation of channels in lipid membranes and the mechanism of ion transport through channels may be studied in experiments with certain hydrophobic peptides such as gramicidin.
Isolated murine skin was burnt in a standard high temperature burn model (250°C, 20 sec). From th... more Isolated murine skin was burnt in a standard high temperature burn model (250°C, 20 sec). From the burnt skin a lipid protein complex was isolated which exhibited toxic activities after intraperitoneal injection into acceptor mice [1]. Metabolic and ultrastructural studies using rat liver perfusion and isolated hepatocytes as target systems revealed alterations of cellular and especially of mitochondrial membranes and a reduced adaptability in gluconeogenesis and urea synthesis after stimulation with amino acids and lactate. After lipid extraction by organic solvents intravenous injection of the apo-protein into rabbits caused platelet aggregation and an increase of activity of lysosomal enzymes in serum.
Republic of Germany * This research was supported by Grants F1 126/3-2, 3-3, SFB 176, Graduierten... more Republic of Germany * This research was supported by Grants F1 126/3-2, 3-3, SFB 176, Graduiertenkolleg Ka 456/5-1, and Ha 1232/8-1 from the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The Tsx protein from the outer membrane of Escherichia coli is known to be involved in the permea... more The Tsx protein from the outer membrane of Escherichia coli is known to be involved in the permeation of nucleosides across the outer membrane under limiting substrate conditions. We purified Tsx from an E. coli strain that overproduces Tsx. The purified protein was still functional since it could neutralize the Tsxspecific bacteriophage T6 in vitro. When the purified
The determinants of the Actinobacillus pleuropneumoniae RTX toxins ApxI, ApxII, and ApxIII were e... more The determinants of the Actinobacillus pleuropneumoniae RTX toxins ApxI, ApxII, and ApxIII were expressed in an Escherichia coli strain. The toxins were concentrated from the supernatants of cell cultures. The addition of the toxins to the aqueous-phase-bathing lipid bilayer membranes resulted in an increase in the membrane conductance when membranes made of asolectin or phosphatidylethanolamine were used. The toxins were relatively inactive in membranes made of other lipids. The membrane activity (i.e., the number of channels formed at a given Apx concentration) was different for each of the three Apx toxins. That of ApxI, which has the strongest cytotoxic activity, was highest, followed by that of ApxIII and ApxII, which is the least cytotoxic. The conductance increases of ApxIII and ApxII were smaller by factors of 10 and 50, respectively, than that of ApxI under otherwise identical conditions. Single-channel experiments demonstrated that all three Apx toxins formed ion-permeable...
Tc toxins are modular toxin systems that are composed of a pentameric membrane translocator (TcA)... more Tc toxins are modular toxin systems that are composed of a pentameric membrane translocator (TcA) and a cocoon (TcB and TcC) encapsulating the toxic enzyme. Binding of Tcs to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. Here, we show in atomic detail an assembled Tc toxin complex from P. luminescens in the membrane. We find that the five TcA protomers conformationally adapt to fit around the cocoon during prepore-to-pore transition. The architecture of the Tc toxin complex also allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Mammalian lipids with zwitterionic head groups are preferred over other lipids for Tc toxin integration. The translocated toxic enzyme, which can be partially visualized, transiently interacts with alternating negative charges and hydrophobic stretches.
Low-molecular weight oligomers of amyloid-b (Ab) peptides have emerged as primary toxic species i... more Low-molecular weight oligomers of amyloid-b (Ab) peptides have emerged as primary toxic species involved in the Alzheimer disease (AD) pathogenesis. Recent findings suggest that oligomers of distinct conformations could self-propagate toward specific aggregate structures. Regarding the generation of specific oligomer conformers, the association of Ab with lipids play an important role. In our laboratory, we investigate the role of membrane lipids and surfactants in modulating Ab aggregation to generate distinct oligomer strains. We hypothesize that such strains that vary in biophysical & biochemical properties, could also lead to specific phenotypes in brains. Growing evidence indicate prion-like progression of Ab aggregate seeds that give rise to specific clinicopatholologic phenotypes in the AD. We investigated the characteristics of Ab oligomers generated in the presence of various ganglioside & anionic phospholipid micelles/liposomes invitro. We found that the Ab peptides interact uniquely with gangliosides and other anionic phospholipids. However, all the oligomers were found to be parallel b-sheet rich with similar molecular weight and hydrodynamic diameter. We also observed distinct range of melting temperatures for all lipid-derived oligomers indicating that they have similar thermodynamic stabilities. Together, these findings indicate that the lipid-derived oligomers may be a part of a distinct class of oligomer strains that could result in a common AD phenotype.
The cationic staphylococcinlike peptide Pep 5 is shown to depolarize bacterial and planar lipid m... more The cationic staphylococcinlike peptide Pep 5 is shown to depolarize bacterial and planar lipid membranes in a voltage-dependent manner. An artificial valinomycin-induced potassium diffusion potential across the cytoplasmic membrane of Staphylococcus cohnii 22 was sufficient to promote Pep 5 action. Thus, evidence is provided that a membrane potential of sufficient magnitude is the only prerequisite for Pep 5 activity. The voltage dependence was elucidated by macroscopic conductance measurements with black lipid membranes. A threshold potential of about -90 to -100 mV, which was deduced from experiments with bacterial cells, could be confirmed. Single pores were resolved which often occur as short-lived bursts and fluctuate among different conductance levels. Pore diameters were calculated ranging from 0.1 to 1 nm. Succinylation of the lysine residues of Pep 5 resulted in prolonged pore lifetimes and maintenance of distinct conductance levels. However, the succinylated peptide requi...
Escherichia coli transports Fe3+ as a ferrichrome complex through the outer membrane in an energy... more 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...
Charge-pulse relaxation studies with the positively charged PV-K + complex (cyclo-(D-Val-L-Pro-L-... more Charge-pulse relaxation studies with the positively charged PV-K + complex (cyclo-(D-Val-L-Pro-L-Val-D-Pro)3) and the negatively charged lipophilic ion dipicrylamine (DPA-) have been performed in order to study the influence of structural properties on ion transport through lipid bilayer membranes. First, the thickness of monoolein membranes was varied over a wide range using different n-alkanes and solvent-free membranes. The thickness (d) of the hydrocarbon core of these membranes varied between 4.9 and 2.5 nm. For both transport systems the partition coefficient/~ was found to be rather insensitive to variations in d. The same was valid for the translocation rate constant kMs of PV-K § whereas a strong increase of the translocation rate constant k i of DPA-with decreasing d was observed. In a further set of experimental conditions the structure of the lipids, such as number and position of the double bonds in the hydrocarbon chain and its chain length as well as the nature of the polar head group, was varied. The translocation constant k~s of PV-K § transport was found to be much more sensitive to these variations than k~ of DPA- .
Mouse liver peroxisomes were isolated by centrifugation in a self-generated Percoll gradient foll... more Mouse liver peroxisomes were isolated by centrifugation in a self-generated Percoll gradient followed by an Optiprep density gradient centrifugation. Peroxisomes contributed 90-96% of the total protein content in the fraction, as confirmed by marker enzyme assays, protein pattern in SDS-PAGE, immunoblotting, and electron microscopy. Solubilized peroxisomal membrane proteins were reconstituted into a planar lipid bilayer. A single-channel conductance monitoring of the reconstituted lipid bilayer revealed the presence of two pore-forming components with a conductance in 1 M KCl of 1.3 nS and 2.5 nS. Control experiments with fractions enriched in mitochondria, lysosomes, and fragments of endoplasmic reticulum showed that the peroxisomal channel-forming activities were not due to admixture of isolated peroxisomes with other cellular organelles. The peroxisomal channels were well preserved in membrane preparations but became unstable after solubilization from the membranes by detergent.
Moraxella catarrhalis is a gram-negative respiratory pathogen that is an important causative agen... more Moraxella catarrhalis is a gram-negative respiratory pathogen that is an important causative agent for otitis media and exacerbations of chronic obstructive pulmonary disease. We have previously predicted the outer membrane protein M35 to be a general porin, and in the current study, we have investigated the function of M35 and its importance for survival of M. catarrhalis in vivo. Lipid bilayer experiments reveal that refolded M35 functions as a channel that is typical of gram-negative bacterial porins. M35 forms wide and water-filled channels with a single-channel conductance of about 1.25 nS in 1 M KCl solution and has only a small selectivity for cations over anions. When the in vitro growth characteristics of two M35 deletion mutant strains of M. catarrhalis were compared to the wild-type parent isolates, the growth of the mutant strains was inhibited only under nutrient-poor conditions. This growth defect could be eliminated by additional glutamic acid, but not additional aspartic acid, glycine, sucrose, or glucose. The mutant strains compensated for the lack of M35 by enhancing their uptake of glutamic acid, and this enhanced rate of glutamic acid uptake was attributed to the compensatory upregulation of a protein of approximately 40 kDa. M35 was also found to be essential for nasal colonization of mice, demonstrating that its presence is essential for survival of M. catarrhalis in vivo. These results suggest that M35 is a general porin that is necessary for the uptake of important energy sources by M. catarrhalis and that it is likely that M35 is an essential functional protein for in vivo colonization.
Publisher Summary This chapter describes the relaxation techniques and noise analysis of ion carr... more Publisher Summary This chapter describes the relaxation techniques and noise analysis of ion carriers in planar bilayers. Only limited information on the carrier system can be obtained from stationary conductance measurements. Relaxation techniques may be used for the evaluation of the rate constants of the single transport steps. In a relaxation experiment an external parameter, such as pressure, temperature, or voltage, is quickly changed; after the perturbation the approach of the system toward a new stationary state is followed. In the analysis of relaxation experiments with alkali ion carders, it is usually assumed that the association and dissociation reactions in the interface are voltage independent and that the only transport step affected by the electric field in the membrane is the translocation of MS + . Analysis of current noise represents an alternative method for evaluating the rate constants of a carrier system. A straightforward method of extracting information from the fluctuating part of the current δI ( t ) consists in measuring the spectral intensity of current noise, because a function of frequency is the value of δI ( t ) 2 per unit frequency interval.
The major outer membrane protein of Rhodobacter capsulatus 37 b4 (capsule-free) was isolated. St... more The major outer membrane protein of Rhodobacter capsulatus 37 b4 (capsule-free) was isolated. Strong porin-activity was observed after reconstitution into artificial lipid bilayer membranes with a single channel conductance of 3.15 nS in Im KC1. The porin migrated as a broad, single band (Mr above 90,000) on sodium dodecyl sulfate polyacrylamide gel electro phoresis and dissociated into a single species of polypeptides (Mr 36,000) on treatment with EDTA (10 mM at 30 °C, 20 min) or by heating (100 °C, 5 min). Analytical ultracentrifugation studies demonstrated the native porin to be a trimer. The monomers chromatofocused as a single, sharp peak on fast performance liquid chromatography and only one band, corre sponding to an isoelectric point of about 4.0, was obtained on isoelectric focusing. Gas-phase sequencing of the 23 N-terminal residues revealed Glu-Val-Lys-Leu-Ser-Gly-Asp-Ala-Arg-Met-Gly-Val-Met-Tyr-Asn-Gly-Asp-Asp-X-Asn-Phe-Ser-Ser.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988
The mechanism of reversible electric breakdown of lipid membranes is studied. The following stage... more The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0,6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.
The sucrose-specific outer membrane parin ScrY of Salmonella typhimurium was isolated from EscMri... more The sucrose-specific outer membrane parin ScrY of Salmonella typhimurium was isolated from EscMrichia coti K-12 strain KS 26 containing the plasmid pPSOI12. The protein was purified to homogeneity by differential extraction of the cell envelope in the presence of the detergents sodium dodecyl sulfate and lauryl (dimethyl)-amine oxide (LDAO). The porin had apparent molecular weights of 58 kDa and 120 kDa for the monomer and for the trimer, respectively, on SDS/PAGE. The purified trimers were crystallized using poly(ethylene glycol) 2000 and the detergents octylglucoside (OG) and hexyl-(dimethyl)-amine oxide (C6DAO). X-ray diffraction of the crystals showed reflections to 2'3 A. The space group of the crystals was R3 and the lattice constants of the hexagonal axes were a = b = 112'85 A and c = 149'9 A. The crystal volume per unit of protein molecular weight was 3'47 A'jDa.
There is now considerable evidence that the ion permeability of biological membranes is not a dif... more There is now considerable evidence that the ion permeability of biological membranes is not a diffuse property of the whole membrane but depends on the presence of specialized components in the membrane. Such a functional component may be a mobile carrier molecule that mediates the transport of ions in a sequence of complexation, translocation, and decomplexation steps. The second limiting case of an ion transport mechanism consists of a porelike channel that offers to the ion a hydrophilic pathway through the apolar core of the membrane. The formation of channels in lipid membranes and the mechanism of ion transport through channels may be studied in experiments with certain hydrophobic peptides such as gramicidin.
Isolated murine skin was burnt in a standard high temperature burn model (250°C, 20 sec). From th... more Isolated murine skin was burnt in a standard high temperature burn model (250°C, 20 sec). From the burnt skin a lipid protein complex was isolated which exhibited toxic activities after intraperitoneal injection into acceptor mice [1]. Metabolic and ultrastructural studies using rat liver perfusion and isolated hepatocytes as target systems revealed alterations of cellular and especially of mitochondrial membranes and a reduced adaptability in gluconeogenesis and urea synthesis after stimulation with amino acids and lactate. After lipid extraction by organic solvents intravenous injection of the apo-protein into rabbits caused platelet aggregation and an increase of activity of lysosomal enzymes in serum.
Republic of Germany * This research was supported by Grants F1 126/3-2, 3-3, SFB 176, Graduierten... more Republic of Germany * This research was supported by Grants F1 126/3-2, 3-3, SFB 176, Graduiertenkolleg Ka 456/5-1, and Ha 1232/8-1 from the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The Tsx protein from the outer membrane of Escherichia coli is known to be involved in the permea... more The Tsx protein from the outer membrane of Escherichia coli is known to be involved in the permeation of nucleosides across the outer membrane under limiting substrate conditions. We purified Tsx from an E. coli strain that overproduces Tsx. The purified protein was still functional since it could neutralize the Tsxspecific bacteriophage T6 in vitro. When the purified
The determinants of the Actinobacillus pleuropneumoniae RTX toxins ApxI, ApxII, and ApxIII were e... more The determinants of the Actinobacillus pleuropneumoniae RTX toxins ApxI, ApxII, and ApxIII were expressed in an Escherichia coli strain. The toxins were concentrated from the supernatants of cell cultures. The addition of the toxins to the aqueous-phase-bathing lipid bilayer membranes resulted in an increase in the membrane conductance when membranes made of asolectin or phosphatidylethanolamine were used. The toxins were relatively inactive in membranes made of other lipids. The membrane activity (i.e., the number of channels formed at a given Apx concentration) was different for each of the three Apx toxins. That of ApxI, which has the strongest cytotoxic activity, was highest, followed by that of ApxIII and ApxII, which is the least cytotoxic. The conductance increases of ApxIII and ApxII were smaller by factors of 10 and 50, respectively, than that of ApxI under otherwise identical conditions. Single-channel experiments demonstrated that all three Apx toxins formed ion-permeable...
Tc toxins are modular toxin systems that are composed of a pentameric membrane translocator (TcA)... more Tc toxins are modular toxin systems that are composed of a pentameric membrane translocator (TcA) and a cocoon (TcB and TcC) encapsulating the toxic enzyme. Binding of Tcs to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. Here, we show in atomic detail an assembled Tc toxin complex from P. luminescens in the membrane. We find that the five TcA protomers conformationally adapt to fit around the cocoon during prepore-to-pore transition. The architecture of the Tc toxin complex also allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Mammalian lipids with zwitterionic head groups are preferred over other lipids for Tc toxin integration. The translocated toxic enzyme, which can be partially visualized, transiently interacts with alternating negative charges and hydrophobic stretches.
Low-molecular weight oligomers of amyloid-b (Ab) peptides have emerged as primary toxic species i... more Low-molecular weight oligomers of amyloid-b (Ab) peptides have emerged as primary toxic species involved in the Alzheimer disease (AD) pathogenesis. Recent findings suggest that oligomers of distinct conformations could self-propagate toward specific aggregate structures. Regarding the generation of specific oligomer conformers, the association of Ab with lipids play an important role. In our laboratory, we investigate the role of membrane lipids and surfactants in modulating Ab aggregation to generate distinct oligomer strains. We hypothesize that such strains that vary in biophysical & biochemical properties, could also lead to specific phenotypes in brains. Growing evidence indicate prion-like progression of Ab aggregate seeds that give rise to specific clinicopatholologic phenotypes in the AD. We investigated the characteristics of Ab oligomers generated in the presence of various ganglioside & anionic phospholipid micelles/liposomes invitro. We found that the Ab peptides interact uniquely with gangliosides and other anionic phospholipids. However, all the oligomers were found to be parallel b-sheet rich with similar molecular weight and hydrodynamic diameter. We also observed distinct range of melting temperatures for all lipid-derived oligomers indicating that they have similar thermodynamic stabilities. Together, these findings indicate that the lipid-derived oligomers may be a part of a distinct class of oligomer strains that could result in a common AD phenotype.
The cationic staphylococcinlike peptide Pep 5 is shown to depolarize bacterial and planar lipid m... more The cationic staphylococcinlike peptide Pep 5 is shown to depolarize bacterial and planar lipid membranes in a voltage-dependent manner. An artificial valinomycin-induced potassium diffusion potential across the cytoplasmic membrane of Staphylococcus cohnii 22 was sufficient to promote Pep 5 action. Thus, evidence is provided that a membrane potential of sufficient magnitude is the only prerequisite for Pep 5 activity. The voltage dependence was elucidated by macroscopic conductance measurements with black lipid membranes. A threshold potential of about -90 to -100 mV, which was deduced from experiments with bacterial cells, could be confirmed. Single pores were resolved which often occur as short-lived bursts and fluctuate among different conductance levels. Pore diameters were calculated ranging from 0.1 to 1 nm. Succinylation of the lysine residues of Pep 5 resulted in prolonged pore lifetimes and maintenance of distinct conductance levels. However, the succinylated peptide requi...
Escherichia coli transports Fe3+ as a ferrichrome complex through the outer membrane in an energy... more 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...
Charge-pulse relaxation studies with the positively charged PV-K + complex (cyclo-(D-Val-L-Pro-L-... more Charge-pulse relaxation studies with the positively charged PV-K + complex (cyclo-(D-Val-L-Pro-L-Val-D-Pro)3) and the negatively charged lipophilic ion dipicrylamine (DPA-) have been performed in order to study the influence of structural properties on ion transport through lipid bilayer membranes. First, the thickness of monoolein membranes was varied over a wide range using different n-alkanes and solvent-free membranes. The thickness (d) of the hydrocarbon core of these membranes varied between 4.9 and 2.5 nm. For both transport systems the partition coefficient/~ was found to be rather insensitive to variations in d. The same was valid for the translocation rate constant kMs of PV-K § whereas a strong increase of the translocation rate constant k i of DPA-with decreasing d was observed. In a further set of experimental conditions the structure of the lipids, such as number and position of the double bonds in the hydrocarbon chain and its chain length as well as the nature of the polar head group, was varied. The translocation constant k~s of PV-K § transport was found to be much more sensitive to these variations than k~ of DPA- .
Mouse liver peroxisomes were isolated by centrifugation in a self-generated Percoll gradient foll... more Mouse liver peroxisomes were isolated by centrifugation in a self-generated Percoll gradient followed by an Optiprep density gradient centrifugation. Peroxisomes contributed 90-96% of the total protein content in the fraction, as confirmed by marker enzyme assays, protein pattern in SDS-PAGE, immunoblotting, and electron microscopy. Solubilized peroxisomal membrane proteins were reconstituted into a planar lipid bilayer. A single-channel conductance monitoring of the reconstituted lipid bilayer revealed the presence of two pore-forming components with a conductance in 1 M KCl of 1.3 nS and 2.5 nS. Control experiments with fractions enriched in mitochondria, lysosomes, and fragments of endoplasmic reticulum showed that the peroxisomal channel-forming activities were not due to admixture of isolated peroxisomes with other cellular organelles. The peroxisomal channels were well preserved in membrane preparations but became unstable after solubilization from the membranes by detergent.
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