The channel-forming proteins we explore include Anthrax protective antigen (from Bacillus anthracis), Epsilon toxin (from Clostridium perfringens), VDAC (Voltage-Dependent Anionic Channel from the outer membrane of mitochondria), OmpF (general bacterial porin from Escherichia coli), LamB (sugar-specific bacterial porin from Escherichia coli), alpha-Hemolysin (toxin from Staphylococcus aureus), OprF (porin from Pseudomonas aeruginosa), Alamethicin (amphiphilic peptide toxin from Trichoderma viride), and Syringomycin E (lipopeptide toxin from Pseudomonas syringae). To study these channels under precisely controlled conditions, we first isolate the channel-forming proteins from their host organisms, purify, and then reconstitute them into planar lipid bilayer membranes. Our main goal is to elucidate the physical principles and molecular mechanisms responsible for metabolite flux regulation under normal and pathological conditions. I. Interactions of multi-charged high-affinity toxin inhibitors with anthrax PA63 pore. Many pathogens use the formation of trans-membrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors. This makes them attractive targets for the discovery of new therapeutic agents. Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of beta-cyclodextrin inhibited anthrax lethal toxin action by blocking the trans-membrane pore formed by the protective antigen (PA) subunit of the toxin. Now, as a next step, we studied physical interactions underlying the high potency of one of the most effective toxin inhibitors, namely, cationic aminopropylthio-beta-cyclodextrin. The design of this inhibitor, the sevenfold symmetrical cyclodextrin molecule chemically modified to add seven positive charges, was guided by the symmetry and predominantly negative charge of the PA63 pore. The protective action of this compound has been demonstrated earlier at both single-molecule and whole-organism levels. In the present study, using noise analysis, statistics of time-resolved single-channel closure events, and multichannel measurements, we found that action of this inhibitor is bimodal. When added to the cis side of the membrane, it blocks the channel reversibly. At high salt concentrations, the blockage of the channel is well described as a two-state Markov process, in which both the on- and off-rates are functions of the salt concentration, whereas the applied voltage affects only the off-rate. At salt concentrations smaller than 1.5 M, the second mode of the derivative action on the channel is discovered: addition of the inhibitor enhances voltage gating, making the closed states of the channel more favorable. The effect depends on the lipid composition of the membrane. II. Biophysical properties of the membrane pore formed by Epsilon toxin. Epsilon toxin is the major virulence factor secreted by Gram-positive, spore-forming anaerobic bacteria Clostridium perfringens types B and D. Epsilon toxin is responsible for a rapidly fatal enterotoxaemia in herbivores when their gastrointestinal tracts are colonized by these bacteria leading to in situ toxin production. Driven by the idea of designing a small-molecule blocker of the Epsilon toxin pore similarly to the cyclodextrin-based potent inhibitors discovered earlier for the anthrax PA63 and alpha-Hemolysin pores, we started with the study of the pores physical properties. As a first step we used poly-(ethylene glycol)s of different molecular weights to probe the pores formed by Epsilon toxin in planar lipid bilayers. We found that the pores are highly asymmetric. The cutoff size of polymers entering the pore through its opening from the cis side, the side of toxin addition, is 500 Da, whereas the cutoff size for the polymers entering from the trans side is 2300 Da. Comparing these characteristic molecular weights with those reported earlier for OmpF porin and the alpha-Hemolysin channel, we estimate the radii of cis and trans openings as 0.4 nm and 1.0 nm, respectively. The simplest geometry corresponding to these findings is that of a truncated cone. The asymmetry of the pore is also confirmed by measurements of the reversal potential at oppositely directed salt gradients. The moderate anionic selectivity of the channel is salted-out more efficiently when the salt concentration is higher at the trans side of the pore. This suggests that the residues carrying the positive charge responsible for the anionic selectivity of the Epsilon toxin pore are not localized at its cis opening but are shifted toward the trans side. III. Physical theory of channel-facilitated metabolite transport. This year we studied new aspects of diffusion in confining geometries in order to further advance the continuum diffusion model of solute dynamics in a membrane channel proposed by us earlier. In many problems of practical and theoretical interest, motion of Brownian particles is spatially constrained. When diffusion occurs in quasi-one-dimensional structures, it is intuitively appealing to introduce an effective one-dimensional description. However, there are limitations of such a description. Combining three-dimensional Brownian dynamics simulations with analytical results obtained by solving one-dimensional equations, we were able to establish a new criterion validating reduction to an effective one-dimensional description. We studied unbiased motion of a point Brownian particle in a tube with corrugated walls made of conical sections of a varying length. Effective one-dimensional description in terms of the generalized Fick-Jacobs equation was used to derive a formula which gives the effective diffusion coefficient of the particle as a function of the geometric parameters of the tube. Comparison with the results of Brownian dynamics simulations allowed us to establish the domain of applicability of both the one-dimensional description and the formula for the effective diffusion coefficient. Our analysis demonstrated that the usually accepted requirements are not sufficient for a successful reduction of the three-dimensional diffusion in a tube of a varying cross section to the effective one-dimensional description. One more factor should be taken into account, namely, the characteristic length of the radius variation should be large enough.
| Rostovtseva, Tatiana K; Gurnev, Philip A; Hoogerheide, David P et al. (2018) Sequence diversity of tubulin isotypes in regulation of the mitochondrial voltage-dependent anion channel. J Biol Chem 293:10949-10962 |
| Berezhkovskii, Alexander M; Bezrukov, Sergey M (2018) Stochastic Gating as a Novel Mechanism for Channel Selectivity. Biophys J 114:1026-1029 |
| Hoogerheide, David P; Gurnev, Philip A; Rostovtseva, Tatiana K et al. (2018) Real-Time Nanopore-Based Recognition of Protein Translocation Success. Biophys J 114:772-776 |
| Berezhkovskii, Alexander M; Bezrukov, Sergey M (2018) Effect of stochastic gating on the flux through a membrane channel: a steady-state approach. J Phys Condens Matter 30:254006 |
| Hoogerheide, David P; Gurnev, Philip A; Rostovtseva, Tatiana K et al. (2017) Mechanism of ?-synuclein translocation through a VDAC nanopore revealed by energy landscape modeling of escape time distributions. Nanoscale 9:183-192 |
| Berezhkovskii, Alexander M; Dagdug, Leonardo; Bezrukov, Sergey M (2017) Mean Direct-Transit and Looping Times as Functions of the Potential Shape. J Phys Chem B 121:5455-5460 |
| Hoogerheide, David P; Noskov, Sergei Y; Jacobs, Daniel et al. (2017) Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes. Proc Natl Acad Sci U S A 114:E3622-E3631 |
| Gurnev, Philip A; Stanley, Christopher B; Aksoyoglu, M Alphan et al. (2017) Poly(ethylene glycol)s in Semidilute Regime: Radius of Gyration in the Bulk and Partitioning into a Nanopore. Macromolecules 50:2477-2483 |
| Gurnev, Philip A; Roark, Torri C; Petrache, Horia I et al. (2017) Cation-Selective Channel Regulated by Anions According to Their Hofmeister Ranking. Angew Chem Int Ed Engl 56:3506-3509 |
| Berezhkovskii, Alexander M; Dagdug, Leonardo; Bezrukov, Sergey M (2017) Bulk-mediated surface transport in the presence of bias. J Chem Phys 147:014103 |
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