Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers.

Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. Dioxolane-linked gA dimers formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of dimerization reaction. The most commonly observed and most stable gramicidin A dimer is the main object of this study. This gramicidin dimer remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers, the linear conductance of single-channel proton currents at different proton concentrations was essentially the same for both natural and gA dimers. g(max) and K(D), obtained from fitting experimental points to a Langmuir adsorption isotherm, were approximately 1500 pS and 300 mM, respectively, for both the natural gA and its dimer. In GMO bilayers, however, proton affinities of gA and the dioxolane-dimer were significantly lower (K(D) of approximately 1 and 1.5 M, respectively), and the g(max) higher (approximately 1750 and 2150 pS, respectively) than in PEPC bilayers. Furthermore, the relationship between single-channel conductance and proton concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channel. However, other factors can also influence closing flickers.     

Role of mitochondrial outer membrane in the uncoupling activity of N-terminally glutamate-substituted gramicidin A

Of a series of gramicidin A (gA) derivatives, we have earlier found the peptide [Glu1]gA exhibiting very low toxicity toward mammalian cells, although dissipating mitochondrial membrane potential with almost the same efficiency as gA. Substitution of glutamate for valine at position 1 of the gA amino acid sequence, which is supposed to interfere with the formation of ion-conducting gA channels via head-to-head dimerization, reduces both channel-forming potency of the peptide in planar lipid bilayer membranes and its photonophoric activity in unilamellar liposomes. Here, we compared [Glu1]gA and gA abilities to cause depolarization of the inner mitochondrial membrane in mitochondria and mitoplasts, the latter lacking the outer mitochondrial membrane. Importantly, much less gA was needed to decrease the membrane potential in mitoplasts than in mitochondria, whereas the depolarizing potency of [Glu1]gA was nearly the same in these systems. Moreover, in multilamellar liposomes, [Glu1]gA exhibited more pronounced protonophoric activity than gA, in contrast to the data for unilamellar liposomes. These results allowed us to conclude that [Glu1]gA has a much higher permeability between adjacent lipid membranes than gA. Therefore, the fraction of peptide molecules, reaching the inner mitochondrial membrane upon the addition to cells, is much higher for [Glu1]gA compared to gА. Under these conditions, the decreased cytotoxicity of [Glu1]gA could be associated with its low efficiency as a channel-former dissipating potassium and sodium ion gradients across plasma membrane. The present study highlighted the role of the ability to permeate among various biological membranes for intracellular efficiency of ionophores.     

Genistein can modulate channel function by a phosphorylation-independent mechanism: importance of hydrophobic mismatch and bilayer mechanics

Genistein, a generic tyrosine kinase inhibitor, has been used extensively as a tool to investigate the possible regulation of membrane function by tyrosine phosphorylation. Genistein, in micromolar concentrations, alters the function of numerous ion channels and other membrane proteins, but only in few cases has it been demonstrated that the changes in membrane protein (ion channel) function are due to changes in a protein's phosphorylation status. The major common denominator characterizing proteins that are modulated by genistein seems to be that they are imbedded into, and span, the bilayer component of the plasma membrane. We therefore explored whether genistein could alter ion channel function by a bilayer-mediated mechanism and examined genistein's effect on gramicidin A (gA) channels in planar phospholipid bilayers. gA channels form by transmembrane dimerization of two nonconducting subunits, and genistein potentiates gA channel activity by increasing the appearance rate and prolonging the lifetime of bilayer-spanning gA dimers. That is, genistein shifts the equilibrium between nonconducting monomers and conducting dimers in favor of the bilayer-spanning dimers; the changes in channel activity therefore cannot be due to changes in bilayer fluidity. To obtain further insights into the mechanism underlying this modulation of gA channel function, we examined the effects of genistein on channels formed by gA analogues that differ in amino acid sequence. For a given channel length, the effects of genistein on gA dimerization do not depend on the specific sequence, or the chirality, of the channel-forming gA analogues. In contrast, when we change the channel length (by decreasing or increasing the number of amino acid residues in the sequence), or the bilayer thickness (by changing methylene groups in the acyl chains), the magnitude of genistein's effect increases with increasing hydrophobic mismatch between the channel length and the bilayer thickness. These results strongly suggest that genistein alters bilayer mechanical properties, which in turn modulates channel function. This bilayer-mediated mechanism is likely to apply to other pharmacological reagents and membrane proteins.     

The cell wall of the pathogenic bacterium Rhodococcus equi contains two channel-forming proteins with different properties

We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA(Req) (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA(Req) has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB(Req) [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.     

ATP transport through a single mitochondrial channel, VDAC, studied by current fluctuation analysis.

The "molecular Coulter counter" concept has been used to study transport of ATP molecules through the nanometer-scale aqueous pore of the voltage-dependent mitochondrial ion channel, VDAC. We examine the ATP-induced current fluctuations and the change in average current through a single fully open channel reconstituted into a planar lipid bilayer. At high salt concentration (1 M NaCl), the addition of ATP reduces both solution conductivity and channel conductance, but the effect on the channel is several times stronger and shows saturation behavior even at 50 mM ATP concentration. These results and simple steric considerations indicate pronounced attraction of ATP molecules to VDAC's aqueous pore and permit us to evaluate the effect of a single ATP molecule on channel conductance. ATP addition also generates an excess noise in the ionic current through the channel. Analysis of this excess noise shows that its spectrum is flat in the accessible frequency interval up to several kilohertz. ATP exchange between the pore and the bulk is fast enough not to display any dispersion at these frequencies. By relating the low-frequency spectral density of the noise to the equilibrium diffusion of ATP molecules in the aqueous pore, we calculate a diffusion coefficient D = (1.6-3.3)10(-11) m2/s. This is one order of magnitude smaller than the ATP diffusion coefficient in the bulk, but it agrees with recent results on ATP flux measurements in multichannel membranes using the luciferin/luciferase method.     

Probes of the conduction process of a voltage-gated Cl- channel from Torpedo electroplax

The open-channel conductance properties of a voltage-gated Cl- channel derived from Torpedo californica electroplax and incorporated into planar bilayers were studied by several approaches. In neutral bilayers the channel conductance saturates with Cl- activity according to a rectangular hyperbolic relation with a half-saturation activity of 75 mM and a maximum conductance of 32 pmho. The observation of identical behavior in charged membranes implies that ions permeating the channel do not sense the surface potential of the bulk membrane. The Cl-:Br- permeability ratio, measured under biionic conditions, is independent of salt concentration. SCN- ion reversibly blocks the channel. The voltage dependence of the block implies the existence of two separate blocking sites within the channel: one accessible from the cis side only (the side to which vesicles are added) and the other accessible from the trans side only. The block at each site is competitive with Cl-. The results are consistent with a single-ion Eyring model of the conduction process in which the ion must traverse three kinetic barriers as it permeates the channel and in which the channel can accommodate at most one ion at a time.     

A large, voltage-dependent channel, isolated from mitochondria by water-free chloroform extraction

We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria. Reconstituted channels showed multiple subconductance levels and were voltage-dependent, showing an increased probability of higher conductance states at voltages near zero. In symmetric 150 mM KCl, the maximal conductance of the channel ranged from 350 pS to 750 pS. For voltages >+/-60 mV, conductance fluctuated in the range of approximately 50- approximately 200 pS. In the presence of a 1:3 gradient of KCl, at pH = 7.4, selectivity periodically switched between different states ranging from weakly anion-selective (V(rev) approximately -15 mV) to ideally cation-selective (V(rev) approximately +29 mV), without a significant change in its conductance. Overall, the diverse, but highly reproducible, channel activity most closely resembled the behavior of the permeability transition pore channel seen in patch-clamp experiments on native mitoplasts. We suggest that the isolated complex may represent the ion-conducting module from the permeability transition pore.     

Covalently linked gramicidin channels: effects of linker hydrophobicity and alkaline metals on different stereoisomers

The direct role of the dioxolane group on the gating and single-channel conductance of different stereoisomers of the dioxolane-linked gramicidin A (gA) channels reconstituted in planar lipid bilayers was investigated. Four different covalently linked gA dimers were synthesized. In two of them, the linker was the conventional dioxolane described previously (SS and RR channels). Two gAs were covalently linked with a novel modified dioxolane group containing a retinal attachment (ret-SS and ret-RR gA dimers). These proteins also formed ion channels in lipid bilayers and were selective for monovalent cations. The presence of the bulky and hydrophobic retinal group immobilizes the dioxolane linker in the bilayer core preventing its rotation into the hydrophilic lumen of the pore. In 1 M HCl the gating kinetics of the SS or RR dimers were indistinguishable from their retinal counterparts; the dwell-time distributions of the open and closed states in the SS and ret-SS were basically the same. In particular, the inactivation of the RR was not prevented by the presence of the retinal group. It is concluded that neither the fast closing events in the SS or RR dimers nor the inactivation of the RR are likely to be a functional consequence of the flipping of the dioxolane inside the pore of the channel. On the other hand, the inactivation of the RR dimer was entirely eliminated when alkaline metals (Cs(+) or K(+)) were the permeating cations in the channel. In fact, the open state of the RR channel became extremely stable, and the gating characteristics of both the SS and RR channels were different from what was seen before with permeating protons. As in HCl, the presence of a retinal in the dioxolane linker did not affect the gating behavior of the SS and RR in Cs(+)- or K(+)-containing solutions. Alternative hypotheses concerning the gating of linked gA dimers are discussed.     

The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.     

Ab initio molecular dynamics study of proton transfer in a polyglycine analog of the ion channel gramicidin A.

Proton transfer in biological systems is thought to often proceed through hydrogen-bonded chains of water molecules. The ion channel, gramicidin A (gA), houses within its helical structure just such a chain. Using the density functional theory based ab initio molecular dynamics Car-Parrinello method, the structure and dynamics of proton diffusion through a polyglycine analog of the gA ion channel has been investigated. In the channel, a proton, which is initially present as hydronium (H3O+), rapidly forms a strong hydrogen bond with a nearest neighbor water, yielding a transient H5O2+ complex. As in bulk water, strong hydrogen bonding of this complex to a second neighbor solvation shell is required for proton transfer to occur. Within gA, this second neighbor shell included not only a channel water molecule but also a carbonyl of the channel backbone. The present calculations suggest a transport mechanism in which a priori carbonyl solvation is a requirement for proton transfer.     

Template-free self-assembling fullerene and lipopeptide conjugates of alamethicin form voltage-dependent ion channels of remarkable stability and activity.

N- and C-terminally modified with fullerene or lipopeptide alamethicin molecules were designed for the formation of template-free, self-assembling, voltage-dependent ion conducting channels. The automated solid phase synthesis of the alamethicin-F30 sequence was performed by in situ fluoride activation on 2-chlorotritylchloride-polystyrene resin and the conjugation with fullerenes-C60 and -C70 was carried out in solution. Voltage-dependent bilayer experiments revealed preferred channel sizes for C-terminal alamethicin F30-fullerene-C60 and -C70 conjugates and higher activity compared with native alamethicin, whereas N-terminally linked fullerene balls destabilize pore formation. C-terminal alamethicin F30-fullerene-C70 conjugates show pore states with remarkably long lifetimes of seconds. C-terminal lipopeptide conjugates of alamethicin were prepared by coupling via short peptide spacers with synthetic tripalmitoyl-S-glyceryl-cysteine. which represents the strong membrane anchoring N-terminus of bacterial lipoprotein. Alamethicin-lipopeptide conjugates exhibit high channel forming activities, whereby they self-assemble and adopt preferred pore states with extremely long lifetimes. The novel membrane modifying peptaibol constructs are valuable lead compounds for developments in sensorics related to transmembrane ion conductance.     

Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide

A COOH-terminal tryptic fragment (Mr approximately equal to 20,000) of colicin E1 has been proposed to contain the membrane channel-forming domain of the colicin molecule. A comparison is made of the conductance properties of colicin E1 and its COOH-terminal fragment in planar bilayer membranes. The macroscopic and single channel properties of colicin E1 and its COOH-terminal tryptic fragment are very similar, if not indistinguishable, implying that the NH2-terminal, two-thirds of the colicin E1 molecule, does not significantly influence its channel properties. The channel-forming activity of both polypeptides is dependent upon the presence of a membrane potential, negative on the trans side of the membrane. The average single channel conductance of colicin E1 and the COOH-terminal fragment is 20.9 +/- 3.9 and 19.1 +/- 2.9 picosiemens, respectively. The rate at which both proteins form conducting channels increases as the pH is lowered from 7 to 5. Both molecules require negatively charged lipids for activity to be expressed, exhibit the same ion selectivity, and rectify the current to the same extent. Both polypeptides associate irreversibly with the membrane in the absence of voltage, but subsequent formation of conducting channels requires a negative membrane potential.     

Principles of nanostructure design with protein building blocks

Currently there is increasing interest in nanostructures and their design. Nanostructure design involves the ability to predictably manipulate the properties of the self-assembly of autonomous units. Autonomous units have preferred conformational states. The units can be synthetic material science-based or derived from functional biological macromolecules. Autonomous biological building blocks with available structures provide an extremely rich and useful resource for design. For proteins, the structural databases contain large libraries of protein molecules and their building blocks with a range of shapes, surfaces, and chemical properties. The introduction of engineered synthetic residues or short peptides into these can expand the available chemical space and enhance the desired properties. Here we focus on the principles of nanostructure design with protein building blocks.     

Purification of gramicidin A.

A simple chromatographic purification of the naturally occurring ion channel-forming pentadecapeptide gramicidin A (gA) is presented. This procedure allows gA to be isolated in gram quantities from the commercially available mixture of isomers after chromatography on silica gel. The gramicidin A obtained in this manner is greater than 95% pure as determined by 1HNMR, HPLC, and amino acid analysis.     

Trimethyloxonium modification of batrachotoxin-activated Na channels alters functionally important protein residues.

The extracellular side of single batrachotoxin-activated voltage-dependent Na channels isolated from rat skeletal muscle membranes incorporated into neutral planar lipid bilayers were treated in situ with the carboxyl methylating reagent, trimethyloxonium (TMO). These experiments were designed to determine whether TMO alters Na channel function by a general through-space electrostatic mechanism or by methylating specific carboxyl groups essential to channel function. TMO modification reduced single-channel conductance by decreasing the maximal turnover rate. Modification increased channel selectivity for sodium ions relative to potassium ions as measured under biionic conditions. TMO modification increased the mu-conotoxin (muCTX) off-rate by three orders of magnitude. Modification did not alter the muCTX on-rate at low ionic strength or Na channel voltage-dependent gating characteristics. These data demonstrate that TMO does not act via a general electrostatic mechanism. Instead, TMO targets protein residues specifically involved in ion conduction, ion selectivity, and muCTX binding. These data support the hypothesis that muCTX blocks open-channel current by physically obstructing the ion channel pore.     

The open state gating mechanism of gramicidin a requires relative opposed monomer rotation and simultaneous lateral displacement

The gating mechanism of the open state of the gramicidin A (gA) channel is studied by using a new Monte Carlo Normal Mode Following (MC-NMF) technique, one applicable even without a target structure. The results demonstrate that the lowest-frequency normal mode (NM) at approximately 6.5 cm(-1) is the crucial mode that initiates dissociation. Perturbing the gA dimer in either direction along this NM leads to opposed, nearly rigid-body rotations of the gA monomers around the central pore axis. Tracking this NM by using the eigenvector-following technique reveals the channel's gating mechanism: dissociation via relative opposed monomer rotation and simultaneous lateral displacement. System evolution along the lowest-frequency eigenvector shows that the large-amplitude motions required for gating (dissociation) are not simple relative rigid-body motions of the monomers. Gating involves coupling intermonomer hydrogen bond breaking, backbone realignment, and relative monomer tilt with complex side chain reorganization at the intermonomer junction.     

Flow-through lipid nanotube arrays for structure-function studies of membrane proteins by solid-state NMR spectroscopy

A novel method for studying membrane proteins in a native lipid bilayer environment by solid-state NMR spectroscopy is described and tested. Anodic aluminum oxide (AAO) substrates with flow-through 175 nm wide and 60-mum-long nanopores were employed to form macroscopically aligned peptide-containing lipid bilayers that are fluid and highly hydrated. We demonstrate that the surfaces of both leaflets of such bilayers are fully accessible to aqueous solutes. Thus, high hydration levels as well as pH and desirable ion and/or drug concentrations could be easily maintained and modified as desired in a series of experiments with the same sample. The method allows for membrane protein NMR experiments in a broad pH range that could be extended to as low as 1 and as high as 12 units for a period of up to a few hours and temperatures as high as 70 degrees C without losing the lipid alignment or bilayers from the nanopores. We demonstrate the utility of this method by a solid-state 19.6 T (17)O NMR study of reversible binding effects of mono- and divalent ions on the chemical shift properties of the Leu(10) carbonyl oxygen of transmembrane pore-forming peptide gramicidin A (gA). We further compare the (17)O shifts induced by binding metal ions to the binding of protons in the pH range from 1 to 12 and find a significant difference. This unexpected result points to a difference in mechanisms for ion and proton conduction by the gA pore. We believe that a large number of solid-state NMR-based studies, including structure-function, drug screening, proton exchange, pH, and other titration experiments, will benefit significantly from the method described here.     

Interaction of Heparins and Dextran Sulfates with a Mesoscopic Protein Nanopore

A mechanism of how polyanions influence the channel formed by Staphylococcus aureus α-hemolysin is described. We demonstrate that the probability of several types of polyanions to block the ion channel depends on the presence of divalent cations and the polyanion molecular weight and concentration. For heparins, a 10-fold increase in molecular weight decreases the half-maximal inhibitory concentration, IC₅₀, nearly 10⁴-fold. Dextran sulfates were less effective at blocking the channel. The polyanions are significantly more effective at reducing the conductance when added to the trans side of this channel. Lastly, the effectiveness of heparins on the channel conductance correlated with their influence on the ζ-potential of liposomes. A model that includes the binding of polyanions to the channel-membrane complex via Ca²⁺-bridges and the asymmetry of the channel structure describes the data adequately. Analysis of the single channel current noise of wild-type and site-directed mutant versions of α-hemolysin channels suggests that a single polyanion enters the pore due to electrostatic forces and physically blocks the ion conduction path. The results might be of interest for pharmacology, biomedicine, and research aiming to design mesoscopic pore blockers.     

Modification of positional distribution of fatty acids in phosphatidylinositol of rabbit neutrophils stimulated with formylmethionyl-leucyl-phenylalanine

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the epsilon-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.     

Modification of the conductance,selectivity and concentration-dependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the ?-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.