@article {390, title = {Engineering Biological Nanopore Approaches toward Protein Sequencing}, journal = {ACS Nano}, volume = {17}, year = {2023}, month = {09}, pages = {16369{\textendash}16395}, abstract = {

Biotechnological innovations have vastly improved the capacity to perform large-scale protein studies, while the methods we have for identifying and quantifying individual proteins are still inadequate to perform protein sequencing at the single-molecule level. Nanopore-inspired systems devoted to understanding how single molecules behave have been extensively developed for applications in genome sequencing. These nanopore systems are emerging as prominent tools for protein identification, detection, and analysis, suggesting realistic prospects for novel protein sequencing. This review summarizes recent advances in biological nanopore sensors toward protein sequencing, from the identification of individual amino acids to the controlled translocation of peptides and proteins, with attention focused on device and algorithm development and the delineation of molecular mechanisms with the aid of simulations. Specifically, the review aims to offer recommendations for the advancement of nanopore-based protein sequencing from an engineering perspective, highlighting the need for collaborative efforts across multiple disciplines. These efforts should include chemical conjugation, protein engineering, molecular simulation, machine-learning-assisted identification, and electronic device fabrication to enable practical implementation in real-world scenarios.

}, isbn = {1936-0851}, doi = {10.1021/acsnano.3c05628}, url = {https://doi.org/10.1021/acsnano.3c05628}, author = {Wei, Xiaojun and Penkauskas, Tadas and Reiner, Joseph E. and Kennard, Celeste and Uline, Mark J. and Wang, Qian and Li, Sheng and Aksimentiev, Aleksei and Robertson, Joseph W. F. and Liu, Chang} } @article {366, title = {Expanding the Molecular Alphabet of DNA-Based Data Storage Systems with Neural Network Nanopore Readout Processing}, journal = {Nano Letters}, year = {2022}, month = {02/2022}, abstract = {

DNA is a promising next-generation data storage medium, but challenges remain with synthesis costs and recording latency. Here, we describe a prototype of a DNA data storage system that uses an extended molecular alphabet combining natural and chemically modified nucleotides. Our results show that MspA nanopores can discriminate different combinations and ordered sequences of natural and chemically modified nucleotides in custom-designed oligomers. We further demonstrate singlemolecule sequencing of the extended alphabet using a neural network architecture that classifies raw current signals generated by Oxford Nanopore sequencers with an average accuracy exceeding 60\% (39{\texttimes} larger than random guessing). Molecular dynamics simulations show that the majority of modified nucleotides lead to only minor perturbations of the DNA double helix. Overall, the extended molecular alphabet may potentially offer a nearly 2-fold increase in storage density and potentially the same order of reduction in the recording latency, thereby enabling new implementations of molecular recorders.

}, doi = {10.1021/acs.nanolett.1c04203}, author = {Tabatabaei, S Kasra and Pham, Bach and Pan, Chao and Liu, Jingqian and Chandak, Shubham and Shorkey, Spencer A and Hernandez, Alvaro G and Aleksei Aksimentiev and Chen, Min and Schroeder, Charles M and Milenkovic, Olgica} } @article {356, title = {The emerging landscape of single-molecule protein sequencing technologies}, journal = {Nature Methods}, volume = {18}, year = {2021}, month = {06/2021}, chapter = {604-617}, abstract = {

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

}, doi = {10.1038/s41592-021-01143-1}, url = {https://doi.org/10.1038/s41592-021-01143-1}, author = {Javier Antonio Alfaro and Peggy Bohl{\"a}nder and Mingjie Dai and Mike Filius and Cecil J. Howard and Xander F. van Kooten and Shilo Ohayon and Adam Pomorski and Sonja Schmid and Aleksei Aksimentiev and Eric V. Anslyn and Georges Bedran and Chan Cao and Mauro Chinappi and Etienne Coyaud and Cees Dekker and Gunnar Dittmar and Nicholas Drachman and Rienk Eelkema and David Goodlett and S{\'e}bastien Hentz and Umesh Kalathiya and Neil L. Kelleher and Ryan T. Kelly and Zvi Kelman and Sung Hyun Kim and Bernhard Kuster and David Rodriguez-Larrea and Stuart Lindsay and Giovanni Maglia and Edward M. Marcotte and John P. Marino and Christophe Masselon and Michael Mayer and Patroklos Samaras and Kumar Sarthak and Lusia Sepiashvili and Derek Stein and Meni Wanunu and Mathias Wilhelm and Peng Yin and Amit Meller and Chirlmin Joo} } @article {346, title = {Electrical unfolding of cytochrome during translocation through a nanopore constriction.}, journal = {Proceedings of the National Academy of Sciences}, volume = {118}, year = {2021}, month = {04/2021}, abstract = {

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome (cyt ) through ultrathin silicon nitride (SiN) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of \~{}30 to 100 MV/m, cyt is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (\~{}ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field-induced deformability.

}, issn = {1091-6490}, doi = {10.1073/pnas.2016262118}, author = {Tripathi, Prabhat and Benabbas, Abdelkrim and Mehrafrooz, Behzad and Yamazaki, Hirohito and Aleksei Aksimentiev and Champion, Paul M and Wanunu, Meni} } @article {317, title = {Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore}, journal = {Nature Biotechnology}, volume = {38}, year = {2020}, month = {02/2020}, pages = {176-181}, chapter = {176}, abstract = {

Efforts to sequence single protein molecules in nanopores have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle molecular differences among all twenty amino acids. Here we report ionic current detection of all twenty proteinogenic amino acids in an aerolysin nanopore with the help of a short polycationic carrier. Application of molecular dynamics simulations revealed that the aerolysin nanopore has a built-in single-molecule trap that fully confines a polycationic carrier-bound amino acid inside the sensing region of the aerolysin. This structural feature means that each amino acid spends sufficient time in the pore for sensitive measurement of the excluded volume of the amino acid. We show that distinct current blockades in wild-type aerolysin can be used to identify 13 of the 20 natural amino acids. Furthermore, we show that chemical modifications, instrumentation advances and nanopore engineering offer a route toward identification of the remaining seven amino acids. These findings may pave the way to nanopore protein sequencing.

Research Highlights

1.\ Tang, Lei. "Sensing proteinogenic amino acids." Nature Methods 17(2):126 (2020) DOI:\ 10.1038/s41592-020-0741-z

2.\ Howorka, Stefan, and Zuzanna S. Siwy. "Reading amino acids in a nanopore." Nature Biotechnology 38(2):159 (2020) DOI:\ 10.1038/s41587-019-0401-y

3.\ Nanopores can identify the amino acids in proteins, the first step to sequencing, Highlight by Illinois news bureau.\ \ 

}, isbn = {1546-1696}, doi = {10.1038/s41587-019-0345-2}, url = {https://doi.org/10.1038/s41587-019-0345-2}, author = {Ouldali, Hadjer and Sarthak, Kumar and Ensslen, Tobias and Piguet, Fabien and Manivet, Philippe and Pelta, Juan and Behrends, Jan C. and Aleksei Aksimentiev and Oukhaled, Abdelghani} } @article {297, title = {Effect of temperature and hydrophilic ratio on the structure of poly(N-vinylcaprolactam)-block-poly(dimethylsiloxane)-block-poly(N-vinylcaprolactam) polymersomes}, journal = {ACS Applied Polymer Materials}, volume = {1}, year = {2019}, month = {04/2019}, pages = {722--736}, chapter = {722}, abstract = {

Nanosized polymeric vesicles (polymersomes) assembled from ABA triblock copolymers of poly(N-vinylcaprolactam)-poly(dimethylsiloxane)-poly(N-vinylcaprolactam) (PVCL-PDMS-PVCL) are a promising platform for biomedical applications, as the temperature-responsiveness of the PVCL blocks enables reversible vesicle shrinkage and permeability of the polymersome shell at elevated temperatures. Herein, we explore the effects of molecular weight, polymer block weight ratios, and temperature on the structure of these polymersomes via electron microscopy, dynamic light scattering, small angle neutron scattering (SANS), and all-atom molecular dynamic methods. We show that the shell structure and overall size of the polymersome can be tuned by varying the hydrophilic (PVCL) weight fraction of the polymer: at room temperature, polymers of smaller hydrophilic ratios form larger vesicles that have thinner shells, whereas polymers with higher PVCL content exhibit interchain aggregation of PVCL blocks within the polymersome shell above 50 {\textdegree}C. Model fitting and model-free analysis of the SANS data reveals that increasing the mass ratio of PVCL to the total copolymer weight from 0.3 to 0.56 reduces the temperature-induced change in vesicle diameter by a factor of 3 while simultaneously increasing the change in shell thickness by a factor of 1.5. Finally, by analysis of the shell structures and overall size of polymersomes with various PVCL weight ratios and those without temperature-dependent polymer components, we bring into focus the mechanism of temperature-triggered drug release reported in a previous study. This work provides new fundamental perspectives on temperature-responsive polymersomes and elucidates important structure-property relationships of their constituent polymers.

}, doi = {10.1021/acsapm.8b00259}, url = {https://pubs.acs.org/doi/pdf/10.1021/acsapm.8b00259}, author = {Yiming Yang and Aaron Alford and Kozlovskaya, Veronika and Zhao, Shidi and Joshi, Himanshu and Kim, Eunjung and Qian, Shuo and Urban, Volker and Cropek, Donald and Aleksei Aksimentiev and Kharlampieva, Eugenia} } @article {295, title = {Electro-Mechanical Conductance Modulation of a Nanopore Using a Removable Gate}, journal = {ACS Nano}, volume = {13}, year = {2019}, month = {02/2019}, pages = {2398--2409}, chapter = {2398}, abstract = {

Ion channels form the basis of information processing in livings cells by facilitating exchange of electrical signals across and along cellular membranes. Applying the same principles to man-made systems requires development of synthetic ion channels that can alter their conductance in response to a variety of external manipulations. By combining single-molecule electrical recordings with all-atom molecular dynamics simulations, we here demonstrate a new hybrid nanopore system that allows for both a stepwise change of its conductance and a non-linear current-voltage dependence. The conductance modulation is realized by using a short flexible peptide gate that carries opposite electric charge at its ends. We show that a constant transmembrane bias can position, and in a later stage remove, the peptide gate right at the most sensitive sensing region of a biological nanopore FraC, thus partially blocking its channel and producing a stepwise change in the conductance. Increasing or decreasing the bias while having the peptide gate trapped in the pore stretches or compresses the peptide within the nanopore, thus modulating its conductance in a non-linear but reproducible manner. We envision a range of applications of this removable-gate nanopore system, e.g. from an element of biological computing circuits to a test bed for probing the elasticity of intrinsically disordered proteins.

}, doi = {10.1021/acsnano.8b09266}, url = {https://doi.org/10.1021/acsnano.8b09266}, author = {Shidi Zhao and Restrepo-Perez, Laura and Maglia, Giovanni and Misha Soskine and Chirlmin Joo and Cees Dekker and Aleksei Aksimentiev} } @article {210, title = {Effect of Cytosine Modifications on DNA Flexibility and Nucleosome Mechanical Stability}, journal = {Nature Communications}, volume = {7}, year = {2016}, pages = {10813}, chapter = {10813}, abstract = {

Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility.

}, doi = {10.1038/ncomms10813}, author = {Thuy Ngo and Jejoong Yoo and Dai, Q and Zhang, Q and He, C and Aleksei Aksimentiev and Taekjip Ha} } @article {15, title = {End-to-end attraction of duplex DNA}, journal = {Nucleic Acids Res}, volume = {40}, year = {2012}, month = {2012 May}, pages = {3812-21}, abstract = {

Recent experiments [Nakata, M. et al., End-to-end stacking and liquid crystal condensation of 6 to 20 basepair DNA duplexes. Science 2007; 318:1276-1279] have demonstrated spontaneous end-to-end association of short duplex DNA fragments into long rod-like structures. By means of extensive all-atom molecular dynamic simulations, we characterized end-to-end interactions of duplex DNA, quantitatively describing the forces, free energy and kinetics of the end-to-end association process. We found short DNA duplexes to spontaneously aggregate end-to-end when axially aligned in a small volume of monovalent electrolyte. It was observed that electrostatic repulsion of 5{\textquoteright}-phosphoryl groups promoted the formation of aggregates in a conformation similar to the B-form DNA double helix. Application of an external force revealed that rupture of the end-to-end assembly occurs by the shearing of the terminal base pairs. The standard binding free energy and the kinetic rates of end-to-end association and dissociation processes were estimated using two complementary methods: umbrella sampling simulations of two DNA fragments and direct observation of the aggregation process in a system containing 458 DNA fragments. We found the end-to-end force to be short range, attractive, hydrophobic and only weakly dependent on the ion concentration. The relation between the stacking free energy and end-to-end attraction is discussed as well as possible roles of the end-to-end interaction in biological and nanotechnological systems.

}, keywords = {DNA, Kinetics, Molecular Dynamics Simulation, Nucleic Acid Conformation}, issn = {1362-4962}, doi = {10.1093/nar/gkr1220}, author = {Christopher Maffeo and Luan, Binquan and Aleksei Aksimentiev} } @article {44, title = {Electric and Electrophoretic Inversion of the DNA Charge in Multivalent Electrolytes}, journal = {Soft Matter}, volume = {6}, year = {2010}, month = {2010 Jan 1}, pages = {243-246}, abstract = {

Counterion-induced inversion of the DNA charge was characterized through extensive molecular dynamics simulations. We observed reversal of the DNA motion in an external electric field upon increasing the concentration of trivalent or quadrivalent counterions. In the case of a divalent electrolyte, inversion of the DNA{\textquoteright}s electric charge was observed at high concentrations of the electrolyte but not reversal of the DNA{\textquoteright} electrophoretic motion. We demonstrate that inversion of the DNA{\textquoteright}s electrophoretic mobility results from a complex interplay of electrostatics and hydrodynamics.

}, issn = {1744-683X}, doi = {10.1039/b917973a}, author = {Luan, Binquan and Aleksei Aksimentiev} } @article {46, title = {The effect of calcium on the conformation of cobalamin transporter BtuB}, journal = {Proteins}, volume = {78}, year = {2010}, month = {2010 Apr}, pages = {1153-62}, abstract = {

BtuB is a beta-barrel membrane protein that facilitates transport of cobalamin (vitamin B12) from the extracellular medium across the outer membrane of Escherichia coli. It is thought that binding of B12 to BtuB alters the conformation of its periplasm-exposed N-terminal residues (the TonB box), which enables subsequent binding of a TonB protein and leads to eventual uptake of B12 into the cytoplasm. Structural studies determined the location of the B12 binding site at the top of the BtuB{\textquoteright}s beta-barrel, surrounded by extracellular loops. However, the structure of the loops was found to depend on the method used to obtain the protein crystals, which-among other factors-differed in calcium concentration. Experimentally, calcium concentration was found to modulate the binding of the B12 substrate to BtuB. In this study, we investigate the effect of calcium ions on the conformation of the extracellular loops of BtuB and their possible role in B12 binding. Using all-atom molecular dynamics, we simulate conformational fluctuations of several X-ray structures of BtuB in the presence and absence of calcium ions. These simulations demonstrate that calcium ions can stabilize the conformation of loops 3-4, 5-6, and 15-16, and thereby prevent occlusion of the binding site. Furthermore, binding of calcium ions to extracellular loops of BtuB was found to enhance correlated motions in the BtuB structure, which is expected to promote signal transduction. Finally, we characterize conformation dynamics of the TonB box in different X-ray structures and find an interesting correlation between the stability of the TonB box structure and calcium binding.

}, keywords = {Amino Acid Sequence, Bacterial Outer Membrane Proteins, Calcium, Escherichia coli Proteins, Lipid Bilayers, Membrane Transport Proteins, Models, Molecular, Molecular Dynamics Simulation, Molecular Sequence Data, Protein Binding, Protein Conformation, Vitamin B 12, Vitamin B Complex}, issn = {1097-0134}, doi = {10.1002/prot.22635}, author = {Luan, Binquan and Carr, Rogan and Caffrey, Martin and Aleksei Aksimentiev} } @article {56, title = {Electro-osmotic screening of the DNA charge in a nanopore}, journal = {Phys Rev E Stat Nonlin Soft Matter Phys}, volume = {78}, year = {2008}, month = {2008 Aug}, pages = {021912}, abstract = {

Extensive all-atom molecular dynamics simulations were performed to characterize the microscopic origin of the force experienced by DNA in a bulk electrolyte and a solid-state nanopore when subject to an external electrostatic field E. The effective screening of the DNA charge was found to originate from the hydrodynamic drag of the electro-osmotic flow that is driven by the motion of counterions along the surface of DNA. We show that the effective driving force F in a nanopore obeys the same law as in a bulk electrolyte: F=ximuE , where xi and mu are the friction coefficient and electrophoretic mobility of DNA, respectively. Using this relationship, we suggest a method for determining the effective driving force on DNA in a nanopore that does not require a direct force measurement.

}, keywords = {DNA, Models, Theoretical, Nanostructures, Osmosis, Static Electricity, Water}, issn = {1539-3755}, author = {Luan, Binquan and Aleksei Aksimentiev} } @article {64, title = {Exploring transmembrane transport through alpha-hemolysin with grid-steered molecular dynamics}, journal = {J Chem Phys}, volume = {127}, year = {2007}, month = {2007 Sep 28}, pages = {125101}, abstract = {

The transport of biomolecules across cell boundaries is central to cellular function. While structures of many membrane channels are known, the permeation mechanism is known only for a select few. Molecular dynamics (MD) is a computational method that can provide an accurate description of permeation events at the atomic level, which is required for understanding the transport mechanism. However, due to the relatively short time scales accessible to this method, it is of limited utility. Here, we present a method for all-atom simulation of electric field-driven transport of large solutes through membrane channels, which in tens of nanoseconds can provide a realistic account of a permeation event that would require a millisecond simulation using conventional MD. In this method, the average distribution of the electrostatic potential in a membrane channel under a transmembrane bias of interest is determined first from an all-atom MD simulation. This electrostatic potential, defined on a grid, is subsequently applied to a charged solute to steer its permeation through the membrane channel. We apply this method to investigate permeation of DNA strands, DNA hairpins, and alpha-helical peptides through alpha-hemolysin. To test the accuracy of the method, we computed the relative permeation rates of DNA strands having different sequences and global orientations. The results of the G-SMD simulations were found to be in good agreement in experiment.

}, keywords = {Bacterial Toxins, Biological Transport, Computer Simulation, DNA, Single-Stranded, Electricity, Electrophysiology, Hemolysin Proteins, Hydrogen-Ion Concentration, Lipid Bilayers, Membranes, Models, Molecular, Molecular Conformation, Permeability, Protein Structure, Secondary, Protons, Static Electricity, Time Factors, Water}, issn = {0021-9606}, doi = {10.1063/1.2770738}, author = {David B Wells and Abramkina, Volha and Aleksei Aksimentiev} } @article {68, title = {The electromechanics of DNA in a synthetic nanopore}, journal = {Biophys J}, volume = {90}, year = {2006}, month = {2006 Feb 1}, pages = {1098-106}, abstract = {

We have explored the electromechanical properties of DNA on a nanometer-length scale using an electric field to force single molecules through synthetic nanopores in ultrathin silicon nitride membranes. At low electric fields, E < 200 mV/10 nm, we observed that single-stranded DNA can permeate pores with a diameter >/=1.0 nm, whereas double-stranded DNA only permeates pores with a diameter >/=3 nm. For pores <3.0 nm diameter, we find a threshold for permeation of double-stranded DNA that depends on the electric field and pH. For a 2 nm diameter pore, the electric field threshold is approximately 3.1 V/10 nm at pH = 8.5; the threshold decreases as pH becomes more acidic or the diameter increases. Molecular dynamics indicates that the field threshold originates from a stretching transition in DNA that occurs under the force gradient in a nanopore. Lowering pH destabilizes the double helix, facilitating DNA translocation at lower fields.

}, keywords = {Biophysics, Computer Simulation, DNA, Electrochemistry, Electromagnetic Fields, Electrophoresis, Electrophoresis, Agar Gel, Hydrogen-Ion Concentration, Ions, Membrane Potentials, Microscopy, Electron, Transmission, Models, Molecular, Nanostructures, Nanotechnology, Nucleic Acid Conformation, Polymerase Chain Reaction, Porosity, Silicon Compounds, Time Factors}, issn = {0006-3495}, doi = {10.1529/biophysj.105.070672}, author = {Jiunn B Heng and Aleksei Aksimentiev and Ho, C and Patrick Marks and Yelena V Grinkova and S Sligar and Klaus Schulten and Gregory Timp} } @article {190, title = {Electrical signatures of single-stranded DNA with single base mutations in a nanopore capacitor}, volume = {17}, year = {2006}, pages = {3160{\textendash}3165}, abstract = {

In this paper, we evaluate the magnitude of the electrical signals produced by DNA translocation through a 1\ nm diameter nanopore in a capacitor membrane with a numerical multi-scale approach, and assess the possibility of resolving individual nucleotides as well as their types in the absence of conformational disorder. We show that the maximum recorded voltage caused by the DNA translocation is about 35\ mV, while the maximum voltage signal due to the DNA backbone is about 30\ mV, and the maximum voltage of a DNA base is about 8\ mV. Signals from individual nucleotides can be identified in the recorded voltage traces, suggesting a 1\ nm diameter pore in a capacitor can be used to accurately count the number of nucleotides in a DNA strand. Furthermore, we study the effect of a single base substitution on the voltage trace, and calculate the differences among the voltage traces due to a single base mutation for the sequences C3AC7, C3CC7, C3GC7 and C3TC7. The calculated voltage differences are in the 5{\textendash}10\ mV range. The calculated maximum voltage caused by the translocation of individual bases varies from 2 to 9\ mV, which is experimentally detectable.

}, doi = {10.1088/0957-4484/17/13/014}, author = {Maria E. Gracheva and Aleksei Aksimentiev and Jean-Pierre Leburton} } @article {75, title = {Extending molecular modeling methodology to study insertion of membrane nanopores}, journal = {Proc Natl Acad Sci U S A}, volume = {101}, year = {2004}, month = {2004 Mar 30}, pages = {4337-8}, keywords = {Biosensing Techniques, Computer Simulation, DNA, Lipid Bilayers, Membrane Proteins, Models, Molecular, Protein Conformation}, issn = {0027-8424}, doi = {10.1073/pnas.0400739101}, author = {Aleksei Aksimentiev and Klaus Schulten} }