@article {399, title = {Conformation-Dependent Hydrogen-Bonding Interactions in a Switchable Artificial Metalloprotein}, journal = {Biochemistry}, year = {2024}, abstract = {

Hydrogen-bonding (H-bonding) interactions in metalloprotein active sites can critically regulate enzyme function. Changes in the protein structure triggered by interplay with substrates, products, and partner proteins are often translated to the metallocofactor by way of specific changes in H-bond networks connected to the active site. However, the complexities of metalloprotein architecture and mechanism often preclude our ability to define the precise molecular interactions giving rise to these intricate regulatory pathways. To address this shortcoming, we have developed conformationally switchable artificial metalloproteins (swArMs) in which allosteric Gln-binding triggers protein conformational changes that impact the microenvironment surrounding an installed metallocofactor. Herein, we report a combined structural, spectroscopic, and computational approach to enhance the conformation-dependent changes in H-bond interactions surrounding the metallocofactor site of a swArM. Structure-informed molecular dynamics simulations were employed to predict point mutations that could enhance active site H-bond interactions preferentially in the Gln-bound\ holo-conformation of the swArM. Testing our predictions via the unique infrared spectral signals associated with the metallocofactor site, we have identified three key residues capable of imparting conformational control over the metallocofactor microenvironment. The resultant swArMs not only model biologically relevant structural regulation but also provide an enhanced Gln-responsive biological probe to be leveraged in future biosensing applications.

}, doi = {10.1021/acs.biochem.4c00209}, url = {https://doi.org/10.1021/acs.biochem.4c00209}, author = {Fatima, Saman and Mehrafrooz, Behzad and Boggs, David G. and Ali, Noor and Singh, Swapnil and Thielges, Megan C. and Bridwell-Rabb, Jennifer and Aksimentiev, Aleksei and Olshansky, Lisa} } @article {397, title = {Dehydrated Biomimetic Membranes with Skinlike Structure and Function}, journal = {ACS Applied Materials \& Interfaces}, volume = {16}, year = {2024}, pages = {20865-20877}, abstract = {

Novel vapor-permeable materials are sought after for applications in protective wear, energy generation, and water treatment. Current impermeable protective materials effectively block harmful agents but trap heat due to poor water vapor transfer. Here we present a new class of materials, vapor permeable dehydrated nanoporous biomimetic membranes (DBMs), based on channel proteins. This application for biomimetic membranes is unexpected as channel proteins and biomimetic membranes were assumed to be unstable under dry conditions. DBMs mimic human skin{\textquoteright}s structure to offer both high vapor transport and small molecule exclusion under dry conditions. DBMs feature highly organized pores resembling sweat pores in human skin, but at super high densities (\>1012\ pores/cm2). These DBMs achieved exceptional water vapor transport rates, surpassing commercial breathable fabrics by up to 6.2 times, despite containing \>2 orders of magnitude smaller pores (1 nm vs \>700 nm). These DBMs effectively excluded model biological agents and harmful chemicals both in liquid and vapor phases, again in contrast with the commercial breathable fabrics. Remarkably, while hydrated biomimetic membranes were highly permeable to liquid water, they exhibited higher water resistances after dehydration at values \>38 times that of commercial breathable fabrics. Molecular dynamics simulations support our hypothesis that dehydration induced protein hydrophobicity increases which enhanced DBM performance. DBMs hold promise for various applications, including membrane distillation, dehumidification, and protective barriers for atmospheric water harvesting materials.

}, doi = {10.1021/acsami.3c19572}, url = {https://doi.org/10.1021/acsami.3c19572}, author = {Oh, Hyeonji and Tu, Yu-Ming and Samineni, Laximicharan and De Respino, Sophie and Mehrafrooz, Behzad and Joshi, Himanshu and Massenburg, Lynnicia and Lopez-Marques, Horacio and Elessawy, Nada and Song, Woochul and Behera, Harekrushna and Dhiman, Raman and Boorla, Veda Sheersh and Kher, Kartik and Lin, Yi-Chih and Maranas, Costas and Aksimentiev, Aleksei and D. Freeman, Benny and Kumar, Manish} } @article {392, title = {Sulfur-Containing Foldamer-Based Artificial Lithium Channels}, journal = {Angewandte Chemie International Edition}, volume = {62}, year = {2023}, pages = {e202305623}, abstract = {

Abstract Unlike many other biologically relevant ions (Na+, K+, Ca2+, Cl-, etc) and protons, whose cellular concentrations are closely regulated by highly selective channel proteins, Li+ ion is unusual in that its concentration is well tolerated over many orders of magnitude and that no lithium-specific channel proteins have so far been identified. While one naturally evolved primary pathway for Li+ ions to traverse across the cell membrane is through sodium channels by competing with Na+ ions, highly sought-after artificial lithium-transporting channels remain a major challenge to develop. Here we show that sulfur-containing organic nanotubes derived from intramolecularly H-bonded helically folded aromatic foldamers of 3.6\ {\r A}\ in hollow cavity diameter could facilitate highly selective and efficient transmembrane transport of Li+ ions, with high transport selectivity factors of 15.3 and 19.9 over Na+ and K+ ions, respectively.

}, keywords = {Foldamers, H-Bonds, Lithium Channels, Organic Nanotubes, Supramolecular Chemistry}, doi = {https://doi.org/10.1002/anie.202305623}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.202305623}, author = {Shen, Jie and R, Deepa and Li, Zhongyan and Oh, Hyeonji and Behera, Harekrushna and Joshi, Himanshu and Kumar, Manish and Aksimentiev, Aleksei and Zeng, Huaqiang} } @article {362, title = {DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity}, journal = {Nano Letters}, volume = {21}, year = {2021}, month = {10/2021}, pages = {8634-8641}, chapter = {8634}, abstract = {

Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches, including optical voltage-sensing dyes and genetically encoded voltage indicators, have evolved. Here, we present a DNA nanotechnology-based system and demonstrate its functionality on liposomes. Using DNA origami, we incorporated and optimized different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we used a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA to connect the nanostructure and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit was read out by fluorescence resonance energy transfer (FRET) changes of single sensors attached to liposomes. A FRET change of \~{}5\% for ΔΨ = 100 mV was observed. The working mechanism of the sensor was rationalized by molecular dynamics simulations. Our approach holds potential for an application as nongenetically encoded membrane sensors.

}, doi = {10.1021/acs.nanolett.1c02584}, url = {https://pubs.acs.org/doi/10.1021/acs.nanolett.1c02584?fig=tgr1\&ref=pdf}, author = {Ochmann, Sarah E and Joshi, Himanshu and Büber, Ece and Franquelim, Henri G and Stegemann, Pierre and Sacc{\`a}, Barbara and Keyser, Ulrich F and Aleksei Aksimentiev and Tinnefeld, Philip} } @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 {331, title = {Tailoring interleaflet lipid transfer with a DNA-based synthetic enzyme}, journal = {Nano Letters}, volume = {20}, year = {2020}, month = {05/2020}, pages = {4306-4311}, chapter = {4306}, abstract = {

Lipid membranes, enveloping all living systems, are of crucial importance, and control over their structure and composition is a highly desirable functionality of artificial structures. However, the rational design of protein-inspired systems is still challenging. Here we have developed a highly functional nucleic acid construct that self-assembles and inserts into membranes, enabling lipid transfer between inner and outer leaflets. By designing the structure to account for interactions between the DNA, its hydrophobic modifications, and the lipids, we successfully exerted control over the rate of interleaflet lipid transfer induced by our DNA-based enzyme. Furthermore, we can regulate the level of lipid transfer by altering the concentration of divalent ions, similar to stimuli-responsive lipid-flipping proteins.

}, keywords = {DNA Nanotechnology, dodecane, lipid flipping, lipid membranes, Molecular dynamics, synthetic ion channel}, doi = {10.1021/acs.nanolett.0c00990}, author = {Sobota, Diana and Joshi, Himanshu and Ohmann, Alexander and Aleksei Aksimentiev and Keyser, Ulrich F} } @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 {306, title = {Controlling aggregation of cholesterol-modified DNA nanostructures}, journal = {Nucleic Acid Research }, volume = {47}, year = {2019}, month = {11/2019}, pages = {11441-11451}, chapter = {11441}, abstract = {

DNA nanotechnology allows for the design of programmable DNA-built nanodevices which controllably interact with biological membranes and even mimic the function of natural membrane proteins. Hydrophobic modifications, covalently linked to the DNA, are essential for targeted interfacing of DNA nanostructures with lipid membranes. However, these hydrophobic tags typically induce undesired aggregation eliminating structural control, the primary advantage of DNA nanotechnology. Here, we study the aggregation of cholesterol-modified DNA nanostructures using a combined approach of non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering, confocal microscopy and atomistic molecular dynamics simulations. We show that the aggregation of cholesterol-tagged ssDNA is sequence-dependent, while for assembled DNA constructs, the number and position of the cholesterol tags are the dominating factors. Molecular dynamics simulations of cholesterol-modified ssDNA reveal that the nucleotides wrap around the hydrophobic moiety, shielding it from the environment. Utilizing this behavior, we demonstrate experimentally that the aggregation of cholesterol-modified DNA nanostructures can be controlled by the length of ssDNA overhangs positioned adjacent to the cholesterol. Our easy-to-implement method for tuning cholesterol-mediated aggregation allows for increased control and a closer structure{\textendash}function relationship of membrane-interfacing DNA constructs - a fundamental prerequisite for employing DNA nanodevices in research and biomedicine.

}, keywords = {cholesterol-modified DNA nanostructures,, DNA Nanotechnology}, doi = {https://doi.org/10.1093/nar/gkz914}, url = {https://doi.org/10.1093/nar/gkz914}, author = {Ohmann, Alexander and G{\"o}pfrich, Kerstin and Joshi, Himanshu and Thompson, Rebecca F and Sobota, Diana and Ranson, Neil A and Aleksei Aksimentiev and Keyser, Ulrich F} } @article {286, title = {A synthetic enzyme built from DNA flips 10$^{7}$ lipids per second in biological membranes}, journal = {Nature Communications}, volume = {9}, year = {2018}, month = {07/2018}, pages = {2426}, abstract = {

Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnology to design a synthetic enzyme that substantially outperforms its biological archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biological membranes by forming a toroidal pore that connects the membrane{\textquoteright}s inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid molecules between the bilayer leaflets, rapidly equilibrating the lipid composition. Through a combination of microscopic simulations and fluorescence microscopy we find the lipid transport rate catalyzed by the DNA nanostructure exceeds 107 molecules per second, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biological enzymes. Furthermore, we show that our DNA-based enzyme can control the composition of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine.

}, keywords = {Computational biophysics, DNA nanostructures, Membrane biophysics, Nanobiotechnology}, doi = {10.1038/s41467-018-04821-5}, url = {http://www.nature.com/articles/s41467-018-04821-5}, author = {Ohmann, Alexander and Li, Chen-Yu and Maffeo, Christopher and Al Nahas, Kareem and Baumann, Kevin N. and G{\"o}pfrich, Kerstin and Yoo, Jejoong and Keyser, Ulrich F. and Aleksei Aksimentiev} } @article {281, title = {Optical voltage sensing using DNA origami}, journal = {Nano Letters}, volume = {18}, year = {2018}, pages = {1962-1971}, abstract = {

We explore the potential of DNA nanotechnology for developing novel optical voltage sensing nano-devices that convert a local change of electric potential into optical signals. As a proof-of-concept of the sensing mechanism, we assembled voltage responsive DNA origami structures labelled with a single pair of FRET dyes. The DNA structures were reversibly immobilised on a nanocapillary tip and underwent controlled structural changes upon application of an electric field. The applied field was monitored through a change in FRET efficiency. By exchanging the position of a single dye, we could tune the voltage sensitivity of our DNA origami structure, demonstrating the flexibility and versatility of our approach. The experimental studies were complemented by coarse-grained simulations that characterised voltage-dependent elastic deformation of the DNA nanostructures and the associated change in the distance between the FRET pair. Our work opens a novel pathway for determining the mechanical properties of DNA origami structures, and highlights potential applications of dynamic DNA nanostructures as voltage sensors.

}, doi = {10.1021/acs.nanolett.7b05354}, url = {https://doi.org/10.1021/acs.nanolett.7b05354}, author = {Hemmig, Elisa Alina and Fitzgerald, Clare and Maffeo, Christopher and Hecker, Lisa and Ochmann, Sarah Elisabeth and Aleksei Aksimentiev and Tinnefeld, Philip and Keyser, Ulrich F} } @article {237, title = {Large-Conductance Transmembrane Porin Made from DNA Origami}, journal = {ACS Nano}, volume = {10}, year = {2016}, month = {08/2016}, pages = {8207--8214}, abstract = {

DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance tenfold compared to previous man-made channels. In our design, nineteen cholesterol-tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane (see equilibration trajectory, require login to nanoHUB). Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance (see ionic current trajectory). All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins{\textquoteright} large ionic conductance.

}, doi = {10.1021/acsnano.6b03759}, author = {Kerstin G{\"o}pfrich* and Chen-Yu Li* and Maria Ricci and Satya Prathyusha Bhamidimarri and Jejoong Yoo and Bertalan Gyenes and Alexander Ohmann and Mathias Winterhalter and Aleksei Aksimentiev and Ulrich F Keyser} } @article {236, title = {Ion Channels Made from a Single Membrane-Spanning DNA Duplex}, journal = {Nano Letters}, volume = {16}, year = {2016}, month = {06/2016}, pages = {4665--4669}, abstract = {

Due to their hollow interior, transmembrane channels are capable of opening up pathways for ions across lipid membranes of living cells. Here, we demonstrate ion conduction induced by a single DNA duplex that lacks a hollow central channel. Decorated with six porpyrin-tags, our duplex is designed to span lipid membranes. Combining electrophysiology measurements with all-atom molecular dynamics simulations, we elucidate the microscopic conductance pathway\ (see\ water channel\ trajectory, require login to nanoHUB). Ions flow (see ion transport\ trajectory)\ at the DNA-lipid interface as the lipid head groups tilt towards the amphiphilic duplex forming a toroidal pore filled with water and ions. Ionic current traces produced by the DNA-lipid channel show well-defined insertion steps, closures and gating similar to those observed for traditional protein channels or synthetic pores. Ionic conductances obtained through simulations and experiments are in excellent quantitative agreement. The conductance mechanism realized here, with the smallest possible DNA-based ion channel, offers a route to design a new class of synthetic ion channels with maximum simplicity.

}, doi = {10.1021/acs.nanolett.6b02039}, author = {Kerstin G{\"o}pfrich and Chen-Yu Li and Iwona Mames and Satya Prathyusha Bhamidimarri and Maria Ricci and Jejoong Yoo and Adam Mames and Alexander Ohmann and Mathias Winterhalter and Eugen Stulz and Aleksei Aksimentiev and Ulrich F Keyser} } @article {14, title = {A Stabilized Finite Element Method for Modified Poisson-Nernst-Planck Equations to Determine Ion Flow Through a Nanopore}, journal = {Commun Comput Phys}, volume = {15}, year = {2014}, month = {01/2014}, pages = {93-125}, abstract = {

The conventional Poisson-Nernst-Planck equations do not account for the finite size of ions explicitly. This leads to solutions featuring unrealistically high ionic concentrations in the regions subject to external potentials, in particular, near highly charged surfaces. A modified form of the Poisson-Nernst-Planck equations accounts for steric effects and results in solutions with finite ion concentrations. Here, we evaluate numerical methods for solving the modified Poisson-Nernst-Planck equations by modeling electric field-driven transport of ions through a nanopore. We describe a novel, robust finite element solver that combines the applications of the Newton{\textquoteright}s method to the nonlinear Galerkin form of the equations, augmented with stabilization terms to appropriately handle the drift-diffusion processes. To make direct comparison with particle-based simulations possible, our method is specifically designed to produce solutions under periodic boundary conditions and to conserve the number of ions in the solution domain. We test our finite element solver on a set of challenging numerical experiments that include calculations of the ion distribution in a volume confined between two charged plates, calculations of the ionic current though a nanopore subject to an external electric field, and modeling the effect of a DNA molecule on the ion concentration and nanopore current.

}, issn = {1815-2406}, doi = {10.4208/cicp.101112.100413a}, author = {Chaudhry, Jehanzeb Hameed and Jeffrey Comer and Aleksei Aksimentiev and Olson, Luke N} }