To investigate large ion channels under precisely controlled conditions, we first isolate the channel-forming proteins from their host organisms, purify, and then reconstitute them into planar lipid membranes. Our main goal is to elucidate the physical principles and molecular mechanisms responsible for metabolite flux regulation under normal and pathological conditions. The channel-forming proteins of interest include Voltage-Dependent Anionic Channel from the outer membrane of mitochondria (VDAC), B-components of Clostridium botulinum C2 and Clostridium perfringens iota toxins, Bacillus anthracis protective antigen, Clostridium perfringens Epsilon toxin, Escherichia coli general porin OmpF and sugar-specific LamB, Staphylococcus aureus alpha-Hemolysin, Pseudomonas aeruginosa OprF, Trichoderma viride Alamethicin, and Pseudomonas syringae lipopeptide toxin Syringomycin E. I. Tailor-made beta-cyclodextrins as broad-spectrum inhibitors of binary pore-forming exotoxins. Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxins consist of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and, after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components. We have demonstrated that a 7-fold symmetrical positively charged beta-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-beta-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We established that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound effectively blocked transmembrane channels formed by the B-components of C2 and iota toxins in planar lipid bilayers in vitro in reconstitution experiments. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component. In our previous work, this beta-cyclodextrin derivative was identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria. II. Regulation of voltage-dependent anionic channel from the outer membrane of mitochondria by dimeric tubulin. In our previous work we have shown that one of the most abundant proteins in the cytosol of the majority of eukaryotic cells, dimeric tubulin, is a potent inhibitor of the voltage-dependent anion channel, VDAC, from the outer mitochondrial membrane. The tubulin-VDAC interaction is seen as reversible transitions of the channel, reconstituted into planar lipid membranes, between its open and tubulin-blocked states. Experiments with isolated mitochondria demonstrated that VDAC-tubulin interaction is functionally important in regulation of mitochondrial respiration. The tubulin-blocked state is still highly ion-conductive (about 40% of the open state conductance in 1 M KCl), which may imply that VDAC inhibition by tubulin is limited by the value of this residual conductance. It is believed, however, that the major role of VDAC is regulation of ATP/ADP exchange and not of the flux of small ions, so what is really important is the effect of tubulin blockage on the nucleotide transport. To assess the functional features of the tubulin-blocked state, we have applied three approaches. We first estimated the change in the characteristic radius of VDAC upon its blockage by tubulin using polymer partitioning into the channel in both open and blocked states. Based on the characteristic molecular weight of the polymer that separates partitioning from exclusion, we concluded that the effective cross-sectional area of the channel is reduced by a factor of two as a result of the blockage. Second, we analyzed the blockage-induced change in the channel small-ion selectivity at salt concentrations close to physiological. We showed that selectivity of the channel reverses its sign: from predominantly anionic selectivity in the open state it shifts to cationic selectivity in the tubulin-blocked one. Third, we estimated ATP partitioning into both open and tubulin-blocked channel. We found that while in the open state the addition of ATP reduces channel conductance, it does not change the conductance of the tubulin-blocked state. We concluded that ATP electrostatically and, at least partially sterically, is excluded from the tubulin-blocked state of VDAC thus establishing the functional role of the VDAC-tubulin interaction in regulation of mitochondrial respiration. III. Physical theory of facilitated metabolite transport: A functional role for transporter isoforms. Many of the nutrients that a cell needs for its functioning, such as sugars, amino acids, nucleotides or organic bases, require specialized transporters to cross the cell membrane. The rapid growth of available information, recently characterized as transporter explosion, has led to creation of the transporter classification system, with division of all transporters into channels and carriers. This year we have focused on the physical principles of optimization of carrier-facilitated transport. In the Human Genome there are 43 distinct families of transport systems that comprise more than 300 isoforms of individual solute carriers. Although the majority of these transport systems is responsible for uptake of specific substrates, a substantial number of transporters are used for uptake of the same solute, and often have an overlapping expression of multiple isoforms that exists in the same cell type. So, the naturally arising question is Why are there so many transporter isoforms?. We offer a possible answer to this question by analyzing the carrier-facilitated transport with the focus on the optimal efficiency of the transporter. We demonstrated that at lower substrate concentrations stronger substrate binding is required, and that the deviations from optimal interaction become more critical as the substrate concentration increases, i.e., higher concentrations necessitate more precise tuning. Thus, uniporters designed to transport the same molecule in the same cell have to be optimized with different amino-acid sequences, with one gene coding for a uniporter protein that functions most efficiently at high solute concentrations, while another gene coding for the one that is most efficient at low concentrations. The existence of multiple transporter isoforms that carry the same molecule is well documented for almost any important substrate. Though this variety of isoforms may seem redundant and, in principle, could be explained by the lack of strong evolutionary pressures to decrease the size of the genome, our analysis now offers a different possibility. We have shown that transporter efficiency is fine-tuned to specific ranges of substrate concentration, so that different isoforms might be tailored accordingly, in order to adjust their amino acid composition for the optimal strength of substrate/transporter interactions and the transition rates between different conformations.

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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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