Publications


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.


A heuristic derived from analysis of the ion channel structural proteome permits the rapid identification of hydrophobic gates

Proceedings of the National Academy of Sciences National Academy of Sciences (2019)

S Rao, K Klesse, P Stansfeld, S Tucker, M Sansom

Ion channel proteins control ionic flux across biological membranes through conformational changes in their transmembrane pores. An exponentially increasing number of channel structures captured in different conformational states are now being determined; however, these newly resolved structures are commonly classified as either open or closed based solely on the physical dimensions of their pore, and it is now known that more accurate annotation of their conductive state requires additional assessment of the effect of pore hydrophobicity. A narrow hydrophobic gate region may disfavor liquid-phase water, leading to local dewetting, which will form an energetic barrier to water and ion permeation without steric occlusion of the pore. Here we quantify the combined influence of radius and hydrophobicity on pore dewetting by applying molecular dynamics simulations and machine learning to nearly 200 ion channel structures. This allows us to propose a simple simulation-free heuristic model that rapidly and accurately predicts the presence of hydrophobic gates. This not only enables the functional annotation of new channel structures as soon as they are determined, but also may facilitate the design of novel nanopores controlled by hydrophobic gates.


Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase

Nucleic Acids Research Oxford University Press (2019) gkz797

A Plochowietz, M Mosayebi, A Cuthbert, H Kaju, J Hohlbein, L Domicevica, PC Biggin, A Kapanidis, J Doye, M Sustarsic, TD Craggs

DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA-Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA-Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1-2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.


Load-dependent adaptation near zero load in the bacterial flagellar motor.

Journal of the Royal Society, Interface 16 (2019) 20190300-

JA Nirody, AL Nord, RM Berry

The bacterial flagellar motor is an ion-powered transmembrane protein complex which drives swimming in many bacterial species. The motor consists of a cytoplasmic 'rotor' ring and a number of 'stator' units, which are bound to the cell wall of the bacterium. Recently, it has been shown that the number of functional torque-generating stator units in the motor depends on the external load, and suggested that mechanosensing in the flagellar motor is driven via a 'catch bond' mechanism in the motor's stator units. We present a method that allows us to measure-on a single motor-stator unit dynamics across a large range of external loads, including near the zero-torque limit. By attaching superparamagnetic beads to the flagellar hook, we can control the motor's speed via a rotating magnetic field. We manipulate the motor to four different speed levels in two different ion-motive force (IMF) conditions. This framework allows for a deeper exploration into the mechanism behind load-dependent remodelling by separating out motor properties, such as rotation speed and energy availability in the form of IMF, that affect the motor torque.


Electrophysiological-mechanical coupling in the neuronal membrane and its role in ultrasound neuromodulation and general anaesthesia

Acta Biomaterialia Elsevier 97 (2019) 116-140

A Jerusalem, Z Al-Rekabi, H Chen, A Ercole, M Malboubi, M Tamayo-Elizalde, L Verhagen, S Contera

The current understanding of the role of the cell membrane is in a state of flux. Recent experiments show that conventional models, considering only electrophysiological properties of a passive membrane, are incomplete. The neuronal membrane is an active structure with mechanical properties that modulate electrophysiology. Protein transport, lipid bilayer phase, membrane pressure and stiffness can all influence membrane capacitance and action potential propagation. A mounting body of evidence indicates that neuronal mechanics and electrophysiology are coupled, and together shape the membrane potential in tight coordination with other physical properties. In this review, we summarise recent updates concerning electrophysiological-mechanical coupling in neuronal function. In particular, we aim at making the link with two relevant yet often disconnected fields with strong clinical potential: the use of mechanical vibrations—ultrasound—to alter the electrophysiogical state of neurons, e.g., in neuromodulation, and the theories attempting to explain the action of general anaesthetics.


CHAP: A Versatile Tool for the Structural and Functional Annotation of Ion Channel Pores.

Journal of molecular biology (2019)

G Klesse, S Rao, MSP Sansom, SJ Tucker

The control of ion channel permeation requires the modulation of energetic barriers or "gates" within their pores. However, such barriers are often simply identified from the physical dimensions of the pore. Such approaches have worked well in the past,but there is now evidence that the unusual behaviour of water within narrow hydrophobic pores can produce an energetic barrier to permeation without requiring steric occlusion of the pathway. Many different ion channels have now been shown to exploit "hydrophobic gating" to regulate ion flow, and it is clear that new tools are required for more accurate functional annotation of the increasing number of ion channel structures becoming available. We have previously shown how molecular dynamics simulations of water can be used as a proxy to predict hydrophobic gates, and we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology.


Tracking antibiotic mechanisms.

Nature reviews. Microbiology 17 (2019) 201-201

OJ Pambos, AN Kapanidis


Simultaneous Tracking of Pseudomonas aeruginosa Motility in Liquid and at the Solid-Liquid Interface Reveals Differential Roles for the Flagellar Stators.

mSystems 4 (2019)

AL Hook, JL Flewellen, J-F Dubern, AM Carabelli, IM Zaid, RM Berry, RD Wildman, N Russell, P Williams, MR Alexander

Bacteria sense chemicals, surfaces, and other cells and move toward some and away from others. Studying how single bacterial cells in a population move requires sophisticated tracking and imaging techniques. We have established quantitative methodology for label-free imaging and tracking of individual bacterial cells simultaneously within the bulk liquid and at solid-liquid interfaces by utilizing the imaging modes of digital holographic microscopy (DHM) in three dimensions (3D), differential interference contrast (DIC), and total internal reflectance microscopy (TIRM) in two dimensions (2D) combined with analysis protocols employing bespoke software. To exemplify and validate this methodology, we investigated the swimming behavior of a Pseudomonas aeruginosa wild-type strain and isogenic flagellar stator mutants (motAB and motCD) within the bulk liquid and at the surface at the single-cell and population levels. Multiple motile behaviors were observed that could be differentiated by speed and directionality. Both stator mutants swam slower and were unable to adjust to the near-surface environment as effectively as the wild type, highlighting differential roles for the stators in adapting to near-surface environments. A significant reduction in run speed was observed for the P. aeruginosa mot mutants, which decreased further on entering the near-surface environment. These results are consistent with the mot stators playing key roles in responding to the near-surface environment.IMPORTANCE We have established a methodology to enable the movement of individual bacterial cells to be followed within a 3D space without requiring any labeling. Such an approach is important to observe and understand how bacteria interact with surfaces and form biofilm. We investigated the swimming behavior of Pseudomonas aeruginosa, which has two flagellar stators that drive its swimming motion. Mutants that had only either one of the two stators swam slower and were unable to adjust to the near-surface environment as effectively as the wild type. These results are consistent with the mot stators playing key roles in responding to the near-surface environment and could be used by bacteria to sense via their flagella when they are near a surface.


Modifying Membrane Morphology and Interactions with DNA Origami Clathrin-Mimic Networks.

ACS nano (2019)

CMA Journot, V Ramakrishna, MI Wallace, AJ Turberfield

We describe the triggered assembly of a bio-inspired DNA origami meshwork on a lipid membrane. DNA triskelia, three-armed DNA origami nanostructures inspired by the membrane-modifying protein clathrin, are bound to lipid mono- and bi-layers using cholesterol anchors. Polymerization of triskelia, triggered by the addition of DNA staples, links triskelion arms to form a mesh. Using transmission electron microscopy, we observe nanoscale local deformation of a lipid monolayer induced by triskelion polymerization that is reminiscent of the formation of clathrin-coated pits. We also show that the polymerization of triskelia bound to lipid bilayers modifies interactions between them, inhibiting the formation of a synapse between giant unilamellar vesicles and a supported lipid bilayer.


Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics

Nucleic Acids Research Oxford University Press (OUP) (0)

NC Robb, AJW te Velthuis, E Fodor, AN Kapanidis


Controlling the bioreceptor spatial distribution at the nanoscale for single molecule counting in microwell arrays

ACS Sensors American Chemical Society (2019)

D Daems, I Rutten, J Bath, H Van Gorp, D Decrop, E Pérez Ruiz, A Turberfield, S De Feyter, J Lammertyn


The power of three spatial dimensions

Nature Reviews Microbiology Springer Science and Business Media LLC (2019)

JH Khoo, HL Miller


Structure and assembly of calcium homeostasis modulator proteins

(2019)

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

Abstract Biological membranes of many tissues and organs contain large-pore channels designed to permeate a wide variety of ions and metabolites. 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), which permeate ions and ATP 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 oligomeric assembly remain unclear. Here, we reveal the first structures of two CALHMs, CALHM1 and CALHM2, by single particle cryo-electron microscopy, which show novel assembly of the four transmembrane helices into channels of 8-mers and 11-mers, 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.


Peptide Assembly Directed and Quantified Using Megadalton DNA Nanostructures.

ACS nano (2019)

J Jin, EG Baker, CW Wood, J Bath, DN Woolfson, AJ Turberfield

In nature, co-assembly of polypeptides, nucleic acids and polysaccharides is used to create functional supramolecular structures. Here we show that DNA nanostructures can be used to template interactions between peptides, and to enable the quantification of multivalent interactions that would otherwise not be observable. Our functional building blocks are DNA-peptide hybrids comprising de novo designed dimeric coiled-coil peptides covalently linked to oligonucleotide tags. These hybrids are incorporated in megadalton DNA origami nanostructures and direct nanostructure association through peptide-peptide interactions. Free and bound nanostructures can be counted directly from electron micrographs allowing estimation of the dissociation constants of the peptides linking them. Results for a single peptide-peptide interaction are consistent with measured solution-phase free energies; DNA nanostructures displaying multiple peptides allow the effects of polyvalency to be probed. This use of DNA nanostructures as identifiers allows the binding strengths of homo- and hetero-dimeric peptide combinations to be measured in a single experiment and gives access to dissociation constants that are too low to be quantified by conventional techniques. The work also demonstrates that hybrid biomolecules can be programmed to achieve spatial organization of complex synthetic biomolecular assemblies.


Recent Advances in Understanding σ70-Dependent Transcription Initiation Mechanisms.

Journal of molecular biology (2019)

A Mazumder, AN Kapanidis

Prokaryotic transcription is one of the most studied biological systems, with relevance to many fields including the development and use of antibiotics, the construction of synthetic gene networks, and the development of many cutting-edge methodologies. Here, we discuss recent structural, biochemical, and single-molecule biophysical studies targeting the mechanisms of transcription initiation in bacteria, including the formation of the open complex, the reaction of initial transcription, and the promoter escape step that leads to elongation. We specifically focus on the mechanisms employed by the RNA polymerase holoenzyme with the housekeeping sigma factor σ70. The recent progress provides answers to long-held questions, identifies intriguing new behaviours, and opens up fresh questions for the field of transcription.


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

Scientific Reports Nature Research 9 (2019) 19473

C Gabbutt, J Seifert, W Shen, 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


Confinement-free wide-field ratiometric tracking of single fluorescent molecules

Biophysical Journal Elsevier BV (2019)

B Gilboa, B Jing, TJ Cui, M Sow, A Plochowietz, A Mazumder, AN Kapanidis


Rapid functionalisation and detection of viruses via a novel Ca2+-mediated virus-DNA interaction

Scientific Reports Nature Research 9 (2019) 16219

A Kapanidis, O Pambos, N Robb, J Taylor, A Kent, B Gilboa


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

(2019)

E Nematian-Ardestani, F Abd-Wahab, F Chatelain, H Sun, M Schewe, T Baukrowitz, S Tucker

ABSTRACT 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 + , NH 4 + , 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 + (IC 50 = 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.

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