Biomedical subjects

Yuri N Antonenko

Publications and source records attributed to Yuri N Antonenko.

7 recordsLinked to original sources

PubMed2003

The structure of BtuB with bound colicin E3 R-domain implies a translocon.

Cellular import of colicin E3 is initiated by the Escherichia coli outer membrane cobalamin transporter, BtuB. The 135-residue 100-A coiled-coil receptor-binding domain (R135) of colicin E3 forms a 1:1 complex with BtuB whose structure at a resolution of 2.75 A is reported. Binding of R135 to the BtuB extracellular surface (DeltaG(o) = -12 kcal mol(-1)) is mediated by 27 residues of R135 near the coiled-coil apex. Formation of the R135-BtuB complex results in unfolding of R135 N- and C-terminal ends, inferred to be important for unfolding of the colicin T-domain. Small conformational changes occur in the BtuB cork and barrel domains but are insufficient to form a translocation channel. The absence of a channel and the peripheral binding of R135 imply that BtuB serves to bind the colicin, and that the coiled-coil delivers the colicin to a neighboring outer membrane protein for translocation, thus forming a colicin translocon. The translocator was concluded to be OmpF from the occlusion of OmpF channels by colicin E3.

Bacterial Outer Membrane Proteins↗

PubMed2003

Cytochrome c decelerates channel kinetics of negatively charged gramicidin due to electrostatic interaction.

The effect of cytochrome c on the kinetic properties of ion channels formed by O-pyromellitylgramicidin (OPg), the negatively charged analogue of gramicidin A (gA), in bilayer lipid membranes was studied by the method of sensitized photoinactivation. The addition of cytochrome c to both sides of the membrane caused substantial deceleration of the photoinactivation kinetics of OPg channels which expose three negative charges to the aqueous phase at both sides of the membrane. By contrast, the gA photoinactivation kinetics was unaltered by the addition of cytochrome c. Based on the sensitivity of the observed effect to the ionic strength of the bathing solution, the cytochrome c-induced deceleration of the OPg photoinactivation kinetics reflecting the increase in the OPg channel lifetime was ascribed to electrostatic interaction of positive charges of cytochrome c with negative charges of OPg that resulted in channel clustering. Formation of clusters of OPg channels was previously inferred to explain the polylysine effect on the OPg channel kinetics. The decelerating effect of cytochrome c on OPg channels was observed only at a high number of OPg channels in the membrane, thus suggesting that the interaction between cytochrome c and the charged transmembrane protein requires sufficiently high negative charge density on the surface of the membrane.

Animals↗

PubMed2003

Structural proton diffusion along lipid bilayers.

For H(+) transport between protein pumps, lateral diffusion along membrane surfaces represents the most efficient pathway. Along lipid bilayers, we measured a diffusion coefficient of 5.8 x 10(-5) cm(2) s(-1). It is too large to be accounted for by vehicle diffusion, considering proton transport by acid carriers. Such a speed of migration is accomplished only by the Grotthuss mechanism involving the chemical exchange of hydrogen nuclei between hydrogen-bonded water molecules on the membrane surface, and the subsequent reorganization of the hydrogen-bonded network. Reconstitution of H(+)-binding sites on the membrane surface decreased the velocity of H(+) diffusion. In the absence of immobile buffers, structural (Grotthuss) diffusion occurred over a distance of 100 micro m as shown by microelectrode aided measurements of the spatial proton distribution in the immediate membrane vicinity and spatially resolved fluorescence measurements of interfacial pH. The efficiency of the anomalously fast lateral diffusion decreased gradually with an increase in mobile buffer concentration suggesting that structural diffusion is physiologically important for distances of approximately 10 nm.

Diffusion↗

PubMed2003

Tandem gramicidin channels cross-linked by streptavidin.

The interaction of biotin-binding proteins with biotinylated gramicidin (gA5XB) was studied by monitoring single-channel activity and sensitized photoinactivation kinetics. It was discovered that the addition of streptavidin or avidin to the bathing solutions of a bilayer lipid membrane (BLM) with incorporated gA5XB induced the opening of a channel characterized by approximately doubled single-channel conductance and extremely long open-state duration. We believe that the deceleration of the photoinactivation kinetics observed here with streptavidin and previously (Rokitskaya, T.I., Y.N. Antonenko, E.A. Kotova, A. Anastasiadis, and F. Separovic. 2000. Biochemistry. 39:13053-13058) with avidin reflects the formation of long-lived channels of this type. Both opening and closing of the double-conductance channels occurred via a transient sub-state of the conductance coinciding with that of the usual single-channel transition. The appearance of the double-conductance channels after the addition of streptavidin was preceded by bursts of fast fluctuations of the current with the open state duration of the individual events of 60 ms. The streptavidin-induced double-conductance channels appeared to be inherent only to the gramicidin analogue with a biotin group linked to the COOH terminus through a long linker arm. Including biotinylated phosphatidylethanolamine into the BLM prevented the formation of the double-conductance channels even with the excess streptavidin. In view of the results obtained here, it is suggested that the double-conductance channel represents a tandem of two neighboring gA5XB channels with their COOH termini being cross-linked by the bound streptavidin at both sides of the BLM. The finding that streptavidin induces the formation of the tandem gramicidin channel comprising two channels functioning in concert is considered to be relevant to the physiologically important phenomenon of ligand-induced receptor oligomerization.

Biotin↗

PubMed2002

Tuning the membrane surface potential for efficient toxin import.

Membrane surface electrostatic interactions impose structural constraints on imported proteins. An unprecedented sensitive dependence on these constraints was seen in the voltage-gated import and channel formation by the C-terminal pore-forming domain of the bacteriocin, colicin E1. At physiological ionic strengths, significant channel current was observed only in a narrow interval of anionic lipid content ([L-]), with the maximum current (I(max)) at 25-30 mol% (dioleoyl)-phosphatidylglycerol ([L-]max) corresponding to a surface potential of the lipid bilayer in the absence of protein, psi(o)max = -60 +/- 5 mV. Higher ionic strength shifted [L-]max to larger values, but psi(o)max remained approximately constant. It is proposed that the channel current (i) increases and (ii) decreases at /psi(o)/ values <55 mV and >65 mV, because of (i) electrostatic interactions needed for effective insertion of the channel polypeptide and (ii) constraints due to electrostatic forces on the flexibility needed for cooperative insertion into the membrane. The loss of flexibility for /psi(o)/ 65 mV was demonstrated by the absence of thermally induced intraprotein distance changes of the bound polypeptide. The anionic lipid content, 25-30 mol%, corresponding to the channel current maxima, is similar to that of the target Escherichia coli cytoplasmic membrane and membranes of mesophilic microorganisms. This suggests that one reason the membrane surface potential is tuned in vivo is to facilitate protein import.

Anions↗

PubMed2002

Membrane dipole potential modulates proton conductance through gramicidin channel: movement of negative ionic defects inside the channel.

The effect of membrane dipole potential on gramicidin channel activity in bilayer lipid membranes (BLMs) was studied. Remarkably, it appeared that proton conductance of gramicidin A (gA) channels responded to modulation of the dipole potential oppositely as compared with gA alkali metal cation conductance. In particular, the addition of phloretin, known to reduce the membrane dipole potential, resulted in a decrease in gA proton conductance, on one hand, and an increase in gA alkali metal conductance, on the other hand, whereas 6-ketocholestanol, the agent raising the membrane dipole potential, provoked an increase in gA proton conductance as opposed to a decrease in the alkali metal cation conductance. The peculiarity of the 6-ketocholestanol effect consisted in its dependence on the H(+) concentration. The experiments with the impermeant dipolar compound, phloridzin, showed that the response of proton transport through gramicidin channels to varying the membrane dipole potential did not change qualitatively if the dipole potential of only one monolayer or both monolayers of the BLM was altered. In contrast to gA proton conductance, the single-channel lifetime changed similarly with varying the membrane dipole potential, regardless of the kind of permeant cations (protons or potassium ions). The results of this study could be tentatively accounted for by an assumption that one of the rate-limiting steps of proton conduction through gramicidin channels represents, in fact, movement of negatively charged species (negative ionic defects) across a membrane.

Anti-Bacterial Agents↗

PubMed2002

Polyanions decelerate the kinetics of positively charged gramicidin channels as shown by sensitized photoinactivation.

The effects of different anionic polymers on the kinetic properties of ionic channels formed by neutral gramicidin A (gA) and its positively charged analogs gramicidin-tris(2-aminoethyl)amine (gram-TAEA) and gramicidin-ethylenediamine (gram-EDA) in a bilayer lipid membrane were studied using a method of sensitized photoinactivation. The addition of Konig's polyanion caused substantial deceleration of the photoinactivation kinetics of gram-TAEA channels, which expose three positive charges to the aqueous phase at both sides of the membrane. In contrast, channels formed of gram-EDA, which exposes one positive charge, and neutral gA channels were insensitive to Konig's polyanion. The effect strongly depended on the nature of the polyanion added, namely: DNA, RNA, polyacrylic acid, and polyglutamic acid were inactive, whereas modified polyacrylic acid induced deceleration of the channel kinetics at high concentrations. In addition, DNA was able to prevent the action of Konig's polyanion. In single-channel experiments, the addition of Konig's polyanion resulted in the appearance of long-lived gram-TAEA channels. The deceleration of the gram-TAEA channel kinetics was ascribed to electrostatic interaction of the polyanion with gram-TAEA that reduces the mobility of gram-TAEA monomers and dimers in the membrane via clustering of channels.

Acrylic Resins↗