41 research outputs found
Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery
To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on âIntracellular Traffic and Transport of Bacterial Protein Toxinsâ, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their âsecond lifeâ in a variety of developing medical and technological applications
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Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery
To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on âIntracellular Traffic and Transport of Bacterial Protein Toxinsâ, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their âsecond lifeâ in a variety of developing medical and technological applications
Conductance of Ideally Cation Selective Channel Depends on Anion Type
poster abstractGramicidin A (gA) is a transmembrane, cation selective ion channel that has been used in many
biophysical studies of lipid bilayers, in particular for investigations of lipid-protein interactions and
membrane electrostatics. In addition, it was found that ionic interactions with neutral lipid membranes
also affect the kinetics of gA channels. Here we report measurements of gA ion-channels for a series of
sodium and potassium salts that show an anion-dependence of gA conductance. We find that gA
conductance varies significantly with the anion type with ClO4 and SCN producing distinctly larger
conductance values than Cl, F, and H2PO4. These results can provide new insights into ion-lipid
membrane interactions and ion channel functions in general
Fungicidal Activities and Mechanisms of Action of Pseudomonas syringae pv. syringae Lipodepsipeptide Syringopeptins 22A and 25A
The plant-associated bacterium Pseudomonas syringae pv. syringae simultaneously produces two classes of metabolites: the small cyclic lipodepsinonapeptides such as the syringomycins and the larger cyclic lipodepsipeptide syringopeptins SP22 or SP25. The syringomycins inhibit a broad spectrum of fungi (but particularly yeasts) by lipid-dependent membrane interaction. The syringopeptins are phytotoxic and inhibitory to Gram-positive bacteria. In this study, the fungicidal activities of two major syringopeptins, SP22A and SP25A, and their mechanisms of action were investigated and compared to those of syringomycin E. SP22A and SP25A were observed to inhibit the fungal yeasts Saccharomyces cerevisiae and Candida albicans although less effectively than syringomycin E. S. cerevisiae mutants defective in ergosterol and sphingolipid biosyntheses were less susceptible to SP22A and SP25A but the relative inhibitory capabilities of SRE vs. SP22A and SP25A were maintained. Similar differences were observed for capabilities to cause cellular K+ and Ca2+ fluxes in S. cerevisiae. Interestingly, in phospholipid bilayers the syringopeptins are found to induce larger macroscopic ionic conductances than syringomycin E but form single channels with similar properties. These findings suggest that the syringopeptins target the yeast plasma membrane, and, like syringomycin E, employ a lipid-dependent channel-forming mechanism of action. The differing degrees of growth inhibition by these lipodepsipeptides may be explained by differences in their hydrophobicities. The more hydrophobic SP22A and SP25A might interact more strongly with the yeast cell wall that would create a selective barrier for their incorporation into the plasma membrane
SOLUTION STRUCTURE OF THE TOXIC E. COLI PEPTIDE, TISB
poster abstractAntibiotics act by interfering in bacterial metabolism. Thus, antibiotics are only effective against metabolically active bacteria while dormant cells are highly tolerant to antibiotics. Such persistent bacterial cells may be the main culprits in chronic infectious diseases resistance to antimicrobial thera-py. In Escherichia coli, expression of a toxic peptide, TisB, sends cells into dormancy by decreasing the proton motive force thus decreasing ATP levels. TisB is a 29 amino acid residue peptide with 70% hydrophobic residues. It has a predicted alpha helical transmembrane domain spanning residues 6 - 28. In membrane channel studies, ion transport is observed with TisB and with some TisB mutants. As a preliminary to combining multi-dimensional NMR spectroscopy with circular dichroism to determine the structure of the TisB membrane ion transport complex in lipid micelles, NMR spectroscopy is used to determine the structure of TisB in ethanol
Parkinson Disease-Associated Protein α-Synuclein Blocks VDAC Providing New Insights into Mitochondrial Toxicity
Physical Properties of the Lipid Diphytanoyl Phosphatidylcholine (DPhPc) used for Ion Channel Measurements
Cation-selective channel is regulated by anions according to their Hofmeister ranking
Specificity of small ions, the Hofmeister ranking, is long-known and has many applications including medicine. Yet it evades consistent theoretical description. Here we study the effect of Hofmeister anions on gramicidin A channels in lipid membranes. Counterintuitively, we find that conductance of this perfectly cation-selective channel increases about two-fold in the H2PO4â<ClââBrââNO3â<ClO4â<SCNâ series. Channel dissociation kinetics show even stronger dependence, with the dwell time increasing ~20-fold. While the conductance can be quantitatively explained by the changes in membrane surface potential due to exclusion of kosmotropes from (or accumulation of chaotropes at) the surface, the kinetics proved to be more difficult to treat. We estimate the effects of changes in the energetics at the bilayer surfaces on the channel dwell time, concluding that the change would have to be greater than typically observed for the Hofmeister effect outside the context of the lipid bilayer., Ion specificity and, in particular, the distinctive effects of anions in salt-induced protein precipitation have been known since the 1880âs, when Franz Hofmeister established the ranking of anions in their ability to regulate egg yolk protein water solubility []. Experimental and theoretical studies have given a detailed empirical picture of the phenomenon, the nature of the ionic interactions with the surfaces leading to the Hofmeister effect is still under debate []. The only consensus is that it cannot be explained by standard theories of electrolytes. For example, bromide is unique in that its salts were recognized as a drug to treat epilepsy a couple of dozen years before Hofmeisterâs studies [] and they are still in use to treat specific types of refractory seizures in children [], but the mechanism of their action remains elusive.,
, Hofmeister effect studied with a nanopore in a neutral lipid membrane. Rather unexpectedly, we find that conductance of a purely cation-selective peptide pore is regulated by anions in correlation with their position in the Hofmeister series. Moreover, the pore conformational dynamics are highly sensitive to the anion species. We relate both effects to preferential depletion of kosmotropic anions (accumulation of chaotropic anions) at the membrane-water interface
Correlations of Specific Ionic Effects using Ion Channels and Surface Charge Measurements
poster abstractSpecific ionic effects, as captured in the Hofmeister series, have been observed in many biological phenomena including protein folding and aggregation and lipid bilayer interactions. Previously we have shown that the Hofmeister effect is present in the activity of gramicidin A channels. In particular, measurements of channel open lifetime and conductance in potassium salts clearly show the existence of two distinct ionic classes that could be identified as kosmotropic and chaotropic. To further investigate this behavior, we have measured the zeta potential of diphytanoyl phosphatidylcholine (DPhPC) liposomes in salt solutions. We observe that anions alter the surface charge of the liposomes depending on the classification of the anion as kosmotropic or chaotropic. Chaotropic anions (SCN-, ClO4-) decrease the surface charge of the liposomes while kosmotropic anions (Cl-, H2PO4-, SO42-) have the opposite effect. These results correlate with our previous studies of cation conductance through gramicidin A channels adding new insight into ionic interactions at the lipid-water interface
The antiarrhythmic compound efsevin directly modulates voltageâdependent anion channel 2 by binding to its inner wall and enhancing mitochondrial Ca2+ uptake
Background and Purpose
The synthetic compound efsevin was recently identified to suppress arrhythmogenesis in models of cardiac arrhythmia, making it a promising candidate for antiarrhythmic therapy. Its activity was shown to be dependent on the voltageâdependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. Here, we investigated the molecular mechanism of the efsevinâVDAC2 interaction.
Experimental Approach
To evaluate the functional interaction of efsevin and VDAC2, we measured currents through recombinant VDAC2 in planar lipid bilayers. Using molecular ligandâprotein docking and mutational analysis, we identified the efsevin binding site on VDAC2. Finally, physiological consequences of the efsevinâinduced modulation of VDAC2 were analysed in HLâ1 cardiomyocytes.
Key Results
In lipid bilayers, efsevin reduced VDAC2 conductance and shifted the channel's open probability towards less anionâselective closed states. Efsevin binds to a binding pocket formed by the inner channel wall and the poreâlining Nâterminal αâhelix. Exchange of amino acids N207, K236 and N238 within this pocket for alanines abolished the channel's efsevinâresponsiveness. Upon heterologous expression in HLâ1 cardiomyocytes, both channels, wildâtype VDAC2 and the efsevinâinsensitive VDAC2AAA restored mitochondrial Ca2+ uptake, but only wildâtype VDAC2 was sensitive to efsevin.
Conclusion and Implications
In summary, our data indicate a direct interaction of efsevin with VDAC2 inside the channel pore that leads to modified gating and results in enhanced SRâmitochondria Ca2+ transfer. This study sheds new light on the function of VDAC2 and provides a basis for structureâaided chemical optimization of efsevin