Sergey M. Bezrukov, PhD, Head, Section on Molecular Transport
Philip A. Gurnev, PhD, Postdoctoral Fellow
Ekaterina M. Nestorovich, PhD, Research Fellow
Tatiana K. Rostovtseva, PhD, Staff Scientist
Elnaz Hassanzadeh, BS, Postbaccalaureate Fellow

We investigate the physical principles of channel-facilitated transport of metabolites and other large solutes across cell and organelle membranes. Large ion channels are not only the gateways of metabolite exchange between different cellular compartments and cells, but they are also recognized as multifunctional membrane receptors and components of many toxins. To study the channels under precisely controlled conditions, we reconstitute channel-forming proteins into planar lipid bilayers. The proteins we work with include anthrax protective antigen (from Bacillus anthracis), 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). Channels formed by these proteins and peptides have aqueous pores of 1 to 2 nm in diameter. We approach these large channels by complementing traditional electrophysiological methods with an original concept of "ion channels as molecular Coulter counters." Specifically, our main focus is to study transport at the level of single molecules. Using this strategy, we elucidate the molecular mechanisms responsible for metabolite flux regulation under normal and pathology conditions.

Blocking anthrax lethal toxin

Nestorovich, Bezrukov; in collaboration with Karginov, Leppla, Moayeri

Bacillus anthracis secretes three polypeptides: lethal factor (LF), edema factor (EF), and protective antigen (PA), which all interact at the surface of mammalian cells to form toxic complexes. LF and EF are enzymes that target substrates within the cytosol; PA provides a heptameric pore to allow LF and EF transport into the cytosol. Other than administration of antibiotics shortly after exposure, there is currently no approved effective treatment for inhalational anthrax. We demonstrate a novel approach to disabling the toxin: high-affinity blockage of the PA pore by a rationally designed low-molecular-weight compound that prevents LF and EF entry into cells. Guided by the seven-fold symmetry and predominantly negative charge of the PA pore, we synthesized small cyclic molecules of seven-fold symmetry: beta-cyclodextrins chemically modified to add seven positive charges. By channel reconstitution and high-resolution conductance recording, we show that per-6-(3-aminopropylthio)-beta-cyclodextrin interacts strongly with the PA pore lumen, blocking PA-induced transport at subnanomolar concentrations. The compound protected RAW 264.7 mouse macrophages from anthrax lethal toxin (LeTx, which consists of PA+LF) cytotoxicity. More important, it completely protected the highly susceptible Fischer F344 rats from LeTx. We expect that this approach will provide the basis for a structure-directed drug discovery program to find new and effective treatments for anthrax.

Monitoring initial stages of phage lambda infection

Gurnev, Bezrukov; in collaboration with Oppenheim, Winterhalter

The recent revival of interest in bacteriophages reflects the emerging possibility of using bacteriophages as promising therapeutic and preserving agents in the control of antibiotic-resistant pathogens such as Streptococcus pyogenes, Staphylococcus aureus, and even Bacillus anthracis. This year, we have been able to observe the first step in bacteriophage infection: the docking of phage lambda to its trimeric receptor LamB, or maltoporin at the single-particle level. Reconstituted into a bilayer lipid membrane, the receptor forms stable water-filled pores that conduct ions and sugars. In the absence of substrate (malto-sugars), the three receptor pores are always open, but they are transiently blocked by added permeating maltohexaose. We find that the phage interacts simultaneously with all three monomers of the maltoporin receptor, modifying the monomers' transport properties. Remarkably, although ionic conductance drops by at most 30 percent upon phage docking, sugar access from the side of phage addition is completely obstructed. The statistics of maltohexaose binding to the phage-receptor complex on the side opposite phage docking show that the monomers of maltoporin still bind sugar independently, with the kinetic constants expected for those of the phage-free receptor. This finding suggests that phage docking does not significantly change the structure of the receptor and that the phage-binding regions are close to, but do not overlap with, the sugar-binding domains of the receptor monomers. Our analysis shows, however, that ion fluxes through the pores of the maltoporin share a new common pathway in the phage-receptor complex.

Studying OprF, a porin from Pseudomonas aeruginosa

Nestorovich, Bezrukov; in collaboration with Nikaido, Sugawara

Pseudomonas aeruginosa is an important bacterial pathogen in Western society. It causes many deaths in hospitalized patients and individuals with cystic fibrosis. The difficulty in treating infections caused by this pathogen is largely a function of the fact that low outer-membrane permeability renders antibiotics ineffective. It was long known that the major nonspecific porin of Pseudomonas aeruginosa's outer membrane, OprF, produces a large channel but that the channel permits only a slow diffusion of various solutes. Using high-resolution size fractionation of OprF and reconstitution techniques, we have provided an explanation of this apparent paradox. We found that OprF exists in the form of two conformers wherein the single-domain conformer represents a small fraction of the total protein population. Moreover, the channels formed by the conformer exist mainly in weakly conductive states and switch to the fully open state for only a short time. Therefore, the low permeability of OprF reported earlier is attributable to two factors: primarily to the paucity of the single-domain conformer in the OprF population and, secondarily, to the predominance of weakly conductive subconformations within the single-domain conformer.

Physics of channel-facilitated metabolite transport

Gurnev, Nestorovich, Bezrukov; in collaboration with Berezhkovskii, Hummer, Krasilnikov

The past year's progress in understanding the physics of channel-facilitated transport resulted in several important findings. Making use of our earlier observation that high salt concentrations promote strong attraction between poly-(ethylene glycol) and the water-filled pore of the alpha-hemolysin channel, we were able to observe the capture and release of a single polymer molecule by the pore as a well-defined reversible step in its small-ion current. As a result, we have identified a new phenomenon wherein binding reaction dynamics are modified by the entropic spring properties of a polymer molecule. In a separate study, we investigated the distribution of direct translocation times for particles passing through membrane channels between two reservoirs. The direct translocation time is a conditional first-passage time defined as the residence time of the particle in the channel while passing directly to the other side of the membrane, i.e., without returning to the reservoir from which it entered. We have shown that, counterintuitively, the distributions of direct translocation times are identical for translocation in both directions, independent of any asymmetry in the potential across the channel and, hence, the translocation probabilities. We have also extended our studies on the constructive role of particle-pore interactions and confirmed our earlier predictions that effective translocation requires an extended potential well, a "binding zone," which we have inferred from experiments with antibiotics of the penicillin group.

COLLABORATORS

Alexander Berezhkovskii, PhD, Division of Computational Bioscience, CIT, NIH, Bethesda, MD
Gerhard Hummer, PhD, Laboratory of Chemical Physics, NIDDK, Bethesda, MD
Vladimir A. Karginov, PhD, Innovative Biologics, Inc., Manassas, VA
Oleg Krasilnikov, PhD, Universidad Federal de Pernambuco, Recife, Brazil
Stephen H. Leppla, PhD, Laboratory of Bacterial Diseases, NIAID, Bethesda, MD
Mahtab Moayeri, PhD, Laboratory of Bacterial Diseases, NIAID, Bethesda, MD
Hiroshi Nikaido, MD, University of California at Berkeley, Berkeley, CA
Amos B. Oppenheim, PhD, Hadassah Medical School, Jerusalem, Israel
Etsuko Sugawara, PhD, University of California at Berkeley, Berkeley, CA
Mathias Winterhalter, PhD, International University Bremen, Bremen, Germany

For further information, contact bezrukos@mail.nih.gov.