Publications


Design, optimization and analysis of large DNA and RNA nanostructures through interactive visualization, editing and molecular simulation

Nucleic Acids Research Oxford University Press (OUP) (2020)

E Poppleton, J Bohlin, M Matthies, S Sharma, F Zhang, P Šulc

<jats:title>Abstract</jats:title> <jats:p>This work seeks to remedy two deficiencies in the current nucleic acid nanotechnology software environment: the lack of both a fast and user-friendly visualization tool and a standard for structural analyses of simulated systems. We introduce here oxView, a web browser-based visualizer that can load structures with over 1 million nucleotides, create videos from simulation trajectories, and allow users to perform basic edits to DNA and RNA designs. We additionally introduce open-source software tools for extracting common structural parameters to characterize large DNA/RNA nanostructures simulated using the coarse-grained modeling tool, oxDNA, which has grown in popularity in recent years and is frequently used to prototype new nucleic acid nanostructural designs, model biophysics of DNA/RNA processes, and rationalize experimental results. The newly introduced software tools facilitate the computational characterization of DNA/RNA designs by providing multiple analysis scripts, including mean structures and structure flexibility characterization, hydrogen bond fraying, and interduplex angles. The output of these tools can be loaded into oxView, allowing users to interact with the simulated structure in a 3D graphical environment and modify the structures to achieve the required properties. We demonstrate these newly developed tools by applying them to design and analysis of a range of DNA/RNA nanostructures.</jats:p>


Correction to 'Bioelectrical understanding and engineering of cell biology'.

Journal of the Royal Society, Interface 17 (2020) 20200435-

Z Schofield, GN Meloni, P Tran, C Zerfass, G Sena, Y Hayashi, M Grant, SA Contera, SD Minteer, M Kim, A Prindle, PRF Rocha, MBA Djamgoz, T Pilizota, PR Unwin, M Asally, OS Soyer


Structure and assembly of calcium homeostasis modulator proteins.

Nat Struct Mol Biol 27 (2020) 150-159

JL Syrjanen, K Michalski, T-H Chou, T Grant, S Rao, N Simorowski, SJ Tucker, N Grigorieff, H Furukawa

The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer's disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore size, lipid accommodation and channel activity.


Assembly and Dynamics of the Bacterial Flagellum.

Annual review of microbiology 74 (2020) 181-200

JP Armitage, RM Berry

The bacterial flagellar motor is the most complex structure in the bacterial cell, driving the ion-driven rotation of the helical flagellum. The ordered expression of the regulon and the assembly of the series of interacting protein rings, spanning the inner and outer membranes to form the ∼45-50-nm protein complex, have made investigation of the structure and mechanism a major challenge since its recognition as a rotating nanomachine about 40 years ago. Painstaking molecular genetics, biochemistry, and electron microscopy revealed a tiny electric motor spinning in the bacterial membrane. Over the last decade, new single-molecule and in vivo biophysical methods have allowed investigation of the stability of this and other large protein complexes, working in their natural environment inside live cells. This has revealed that in the bacterial flagellar motor, protein molecules in both the rotor and stator exchange with freely circulating pools of spares on a timescale of minutes, even while motors are continuously rotating. This constant exchange has allowed the evolution of modified components allowing bacteria to keep swimming as the viscosity or the ion composition of the outside environment changes.


A lower X-gate in TASK channels traps inhibitors within the vestibule

Nature (2020)

KEJ Rödström, AK Kiper, W Zhang, S Rinné, ACW Pike, M Goldstein, LJ Conrad, M Delbeck, MG Hahn, H Meier, M Platzk, A Quigley, D Speedman, L Shrestha, SMM Mukhopadhyay, NA Burgess-Brown, SJ Tucker, T Müller, N Decher, EP Carpenter

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. TWIK-related acid-sensitive potassium (TASK) channels—members of the two pore domain potassium (K2P) channel family—are found in neurons1, cardiomyocytes2–4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8–11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12–15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate—which we designate as an ‘X-gate’—created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.


Induced Polarization in MD Simulations of the 5HT3 Receptor Channel

Journal of the American Chemical Society American Chemical Society (2020)

S Tucker, M Sansom

Ion channel proteins form water-filled nanoscale pores within lipid bilayers and their properties are dependent on the complex behavior of water in a nano-confined environment. Using a simplified model of the pore of the 5HT3 receptor (5HT3R) which restrains the backbone structure to that of the parent channel protein from which it is derived we compare additive with polarizable models in describing the behavior of water in nanopores. Molecular Dynamics simulations were performed with four conformations of the channel: two closed state structures, an intermediate state, and an open state, each embedded in a phosphatidylcholine bilayer. Water density profiles revealed that for all water models, the closed and intermediate states exhibited strong dewetting within the central hydrophobic gate region of the pore. However, the open state conformation exhibited varying degrees of hydration, ranging from partial wetting for the TIP4P/2005 water model, to complete wetting for the polarizable AMOEBA14 model. Water dipole moments calculated using polarizable force fields also revealed that water molecules remaining within dewetted sections of the pore resemble gas phase water. Free energy profiles for Na+ and for Cl- ions within the open state pore revealed more rugged energy landscapes using polarizable force fields, and the hydration number profiles of these ions were also sensitive to induced polarization resulting in a substantive reduction of the number of waters within the first hydration shell of Cl- whilst it permeates the pore. These results demonstrate that induced polarization can influence the complex behavior of water and ions within nanoscale pores and provides important new insights into their chemical properties.


Electric Field Induced Wetting of a Hydrophobic Gate in a Model Nanopore Based on the 5-HT3 Receptor Channel

bioRxiv (2020)

G Klesse, S Tucker, MSP Sansom

Abstract In this study we examined the influence of a transmembrane voltage on the hydrophobic gating of nanopores using molecular dynamics simulations. We observed electric field induced wetting of a hydrophobic gate in a biologically inspired model nanopore based on the 5-HT 3 receptor in its closed state, with a field of at least ∼100 mV nm −1 was required to hydrate the pore. We also found an unequal distribution of charged residues can generate an electric field intrinsic to the nanopore which, depending on its orientation, can alter the effect of the external field, thus making the wetting response asymmetric. This wetting response could be described by a simple model based on water surface tension, the volumetric energy contribution of the electric field, and the influence of charged amino acids lining the pore. Finally, the electric field response was used to determine time constants characterising the phase transitions of water confined within the nanopore, revealing liquid-vapour oscillations on a time scale of ~5 ns. This time scale was largely independent of the water model employed and was similar for different sized pores representative of the open and closed states of the pore. Furthermore, our finding that the threshold voltage required for hydrating a hydrophobic gate depends on the orientation of the electric field provides an attractive perspective for the design of rectifying artificial nanopores. ToC/Abstract Graphic <jats:fig id="ufig1" position="float" orientation="portrait" fig-type="figure"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="114157v1_ufig1" position="float" orientation="portrait" />


Altered functional properties of a missense variant in the TRESK K+ channel (KCNK18) associated with migraine and intellectual disability.

Pflugers Archiv : European journal of physiology 472 (2020) 923-930

P Imbrici, E Nematian-Ardestani, S Hasan, M Pessia, SJ Tucker, MC D'Adamo

Mutations in the KCNK18 gene that encodes the TRESK K2P potassium channel have previously been linked with typical familial migraine with aura. Recently, an atypical clinical case has been reported in which a male individual carrying the p.Trp101Arg (W101R) missense mutation in the KCNK18 gene was diagnosed with intellectual disability and migraine with brainstem aura. Here we report the functional characterization of this new missense variant. This mutation is located in a highly conserved residue close to the selectivity filter, and our results show although these mutant channels retain their K+ selectivity and calcineurin-dependent regulation, the variant causes an overall dramatic loss of TRESK channel function as well as an initial dominant-negative effect when co-expressed with wild-type channels in Xenopus laevis oocytes. The dramatic functional consequences of this mutation thereby support a potentially pathogenic role for this variant and provide further insight into the relationship between the structure and function of this ion channel.


Reconfigurable T‐junction DNA origami

Angewandte Chemie Wiley (2020) ange.202006281

K Young, B Najafi, W Sant, S Contera, A Louis, J Doye, A Turberfield, J Bath


Publisher Correction: Structure and assembly of calcium homeostasis modulator proteins.

Nature structural & molecular biology 27 (2020) 305-

JL Syrjanen, K Michalski, T-H Chou, T Grant, S Rao, N Simorowski, SJ Tucker, N Grigorieff, H Furukawa

An amendment to this paper has been published and can be accessed via a link at the top of the paper.


Biophysical characterization of DNA origami nanostructures reveals inaccessibility to intercalation binding sites

Nanotechnology IOP Publishing (2020)

H Miller, KE Dunn, S Schroeter, S Contera, A Wollman, A Hirst, M Leake, D O'Connell


Bioelectrical understanding and engineering of cell biology

Journal of The Royal Society Interface The Royal Society 17 (2020) 20200013

Z Schofield, GN Meloni, P Tran, C Zerfass, G Sena, Y Hayashi, M Grant, SA Contera, SD Minteer, M Kim, A Prindle, P Rocha, MBA Djamgoz, T Pilizota, PR Unwin, M Asally, OS Soyer

The last five decades of molecular and systems biology research have provided unprecedented insights into the molecular and genetic basis of many cellular processes. Despite these insights, however, it is arguable that there is still only limited predictive understanding of cell behaviours. In particular, the basis of heterogeneity in single-cell behaviour and the initiation of many different metabolic, transcriptional or mechanical responses to environmental stimuli remain largely unexplained. To go beyond the status quo, the understanding of cell behaviours emerging from molecular genetics must be complemented with physical and physiological ones, focusing on the intracellular and extracellular conditions within and around cells. Here, we argue that such a combination of genetics, physics and physiology can be grounded on a bioelectrical conceptualization of cells. We motivate the reasoning behind such a proposal and describe examples where a bioelectrical view has been shown to, or can, provide predictive biological understanding. In addition, we discuss how this view opens up novel ways to control cell behaviours by electrical and electrochemical means, setting the stage for the emergence of bioelectrical engineering.


Electric Field Induced Wetting of a Hydrophobic Gate in a Model Nanopore Based on the 5-HT3 Receptor Channel

ACS Nano American Chemical Society (ACS) (2020) acsnano.0c04387

G Klesse, SJ Tucker, MSP Sansom


Design of hidden thermodynamic driving for non-equilibrium systems via mismatch elimination during DNA strand displacement

Nature Communications Springer Nature 11 (2020) 2562

NEC Haley, TE Ouldridge, I Mullor Ruiz, A Geraldini, A Louis, J Bath, AJ Turberfield

Recent years have seen great advances in the development of synthetic self-assembling molecular systems. Designing out-of-equilibrium architectures, however, requires a more subtle control over the thermodynamics and kinetics of reactions. We propose a mechanism for enhancing the thermodynamic drive of DNA strand-displacement reactions whilst barely perturbing forward reaction rates: the introduction of mismatches within the initial duplex. Through a combination of experiment and simulation, we demonstrate that displacement rates are strongly sensitive to mismatch location and can be tuned by rational design. By placing mismatches away from duplex ends, the thermodynamic drive for a strand-displacement reaction can be varied without significantly affecting the forward reaction rate. This hidden thermodynamic driving motif is ideal for the engineering of non-equilibrium systems that rely on catalytic control and must be robust to leak reactions.


Selectivity filter instability dominates the low intrinsic activity of the TWIK-1 K2P K+ Channel.

The Journal of biological chemistry (2019)

E Nematian-Ardestani, MF Abd-Wahab, FC Chatelain, H Sun, M Schewe, T Baukrowitz, SJ Tucker

Two-pore domain (K2P) K+ channels have many important physiological functions. However, the functional properties of the TWIK-1 (K2P1.1/KCNK1) K2P channel remain poorly characterized because heterologous expression of this ion channel yields only very low levels of functional activity. Several underlying reasons have been proposed, including TWIK-1 retention in intracellular organelles, inhibition by post-translational sumoylation, a hydrophobic barrier within the pore, and a low open probability of the selectivity filter (SF) gate. By evaluating these various potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating the underlying mechanism, we observed that the low activity of the SF gate appears to arise from the inefficiency of K+ in stabilizing an active (i.e. conductive) SF conformation. In contrast, other permeant ion species, such as Rb+, NH4+, and Cs+, strongly promoted a pH-dependent activated conformation. Furthermore, many K2P channels are activated by membrane depolarization via a SF-mediated gating mechanism, but we found here that only very strong, non-physiological depolarization produces voltage-dependent activation of heterologously expressed TWIK-1. Remarkably, we also observed that TWIK-1 Rb+ currents are potently inhibited by intracellular K+ (IC50 = 2.8 mM). We conclude that TWIK-1 displays unique SF gating properties among the family of K2P channels. In particular, the apparent instability of the conductive conformation of the TWIK-1 SF in the presence of K+ appears to dominate the low levels of intrinsic functional activity observed when the channel is expressed at the cell surface.


Induced Polarization in MD Simulations of the 5HT3 Receptor Channel

(2020)

G Klesse, S Rao, S Tucker, MSP Sansom

Abstract Ion channel proteins form water-filled nanoscale pores within lipid bilayers and their properties are dependent on the complex behavior of water in a nano-confined environment. Using the pore of the 5HT3 receptor (5HT3R) we compare additive with polarizable models in describing the behavior of water in nanopores. Molecular Dynamics simulations were performed with four conformations of the channel: two closed state structures, an intermediate state, and an open state, each embedded in a phosphatidylcholine bilayer. Water density profiles revealed that for all water models, the closed and intermediate states exhibited strong dewetting within the central hydrophobic gate region of the pore. However, the open state conformation exhibited varying degrees of hydration, ranging from partial wetting for the TIP4P/2005 water model, to complete wetting for the polarizable AMOEBA14 model. Water dipole moments calculated using polarizable force fields also revealed that water molecules remaining within dewetted sections of the pore resemble gas phase water. Free energy profiles for Na+ and for Cl− ions within the open state pore revealed more rugged energy landscapes using polarizable force fields, and the hydration number profiles of these ions were also sensitive to induced polarization resulting in a substantive reduction of the number of waters within the first hydration shell of Cl− whilst it permeates the pore. These results demonstrate that induced polarization can influence the complex behavior of water and ions within nanoscale pores and provides important new insights into their chemical properties. ToC Graphic <jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="971853v1_ufig1" position="float" orientation="portrait" />


Annotating Ion Channel Pores: Structures, Hydrophobicity and the Threshold for Permeation

BIOPHYSICAL JOURNAL 118 (2020) 272A-272A

S Rao, G Klesse, SJ Tucker, MSP Sansom


Induced Polarization in Molecular Dynamics Simulations of the 5-HT3 Receptor Channel.

J Am Chem Soc 142 (2020) 9415-9427

G Klesse, S Rao, SJ Tucker, MSP Sansom

Ion channel proteins form water-filled nanoscale pores within lipid bilayers, and their properties are dependent on the complex behavior of water in a nanoconfined environment. Using a simplified model of the pore of the 5-HT3 receptor (5HT3R) which restrains the backbone structure to that of the parent channel protein from which it is derived, we compare additive with polarizable models in describing the behavior of water in nanopores. Molecular dynamics simulations were performed with four conformations of the channel: two closed state structures, an intermediate state, and an open state, each embedded in a phosphatidylcholine bilayer. Water density profiles revealed that for all water models, the closed and intermediate states exhibited strong dewetting within the central hydrophobic gate region of the pore. However, the open state conformation exhibited varying degrees of hydration, ranging from partial wetting for the TIP4P/2005 water model to complete wetting for the polarizable AMOEBA14 model. Water dipole moments calculated using polarizable force fields also revealed that water molecules remaining within dewetted sections of the pore resemble gas phase water. Free energy profiles for Na+ and for Cl- ions within the open state pore revealed more rugged energy landscapes using polarizable force fields, and the hydration number profiles of these ions were also sensitive to induced polarization resulting in a substantive reduction of the number of waters within the first hydration shell of Cl- while it permeates the pore. These results demonstrate that induced polarization can influence the complex behavior of water and ions within nanoscale pores and provides important new insights into their chemical properties.


AFM nanoindentation reveals decrease of elastic modulus of lipid bilayers near freezing point of water

Scientific Reports Nature Research 9 (2019) 19473

C Gabbutt, W Shen, J Seifert, S Antoranz Contera


Polymeric microellipsoids with programmed magnetic 2 anisotropy for controlled rotation using low (≈10 mT) 3 magnetic fields

Applied Materials Today Elsevier 18 (2019) 100511

A Bonilla Brunner, I Llorente Garcia, B Jang, M Amano Patino, V Alimchandani, BJ Nelson, S Pane, S Antoranz Contera

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