Publications


Structural Mechanisms of Mechanosensitivity in the TREK-2 K2P Potassium Channel

(2017)

SJ Tucker


The structural movement of the TM4 segment during pore gating in TREK1 channels

(2017)

F Schulz, M Rapedius, SJ Tucker, T Baukrowitz


A conserved drug-binding site controls the selectivity filter gate in K2P K+ channels

(2017)

M Schewer, F Schulz, U Mert, H Sun, T Koehler, M Tegtmeier, M Musinszki, H Belabed, M Nazare, SJ Tucker, T Baukrowitz


The effects of stretch activation on ionic selectivity of the TREK-2 K2P K+ channel.

Channels (Austin, Tex.) 11 (2017) 482-486

E Nematian-Ardestani, V Jarerattanachat, P Aryal, MSP Sansom, SJ Tucker

The TREK-2 (KCNK10) K2P potassium channel can be regulated by variety of polymodal stimuli including pressure. In a recent study, we demonstrated that this mechanosensitive K+ channel responds to changes in membrane tension by undergoing a major structural change from its 'down' state to the more expanded 'up' state conformation. These changes are mostly restricted to the lower part of the protein within the bilayer, but are allosterically coupled to the primary gating mechanism located within the selectivity filter. However, any such structural changes within the filter also have the potential to alter ionic selectivity and there are reports that some K2Ps, including TREK channels, exhibit a dynamic ionic selectivity. In this addendum to our previous study we have therefore examined whether the selectivity of TREK-2 is altered by stretch activation. Our results reveal that the filter remains stable and highly selective for K+ over Na+ during stretch activation, and that permeability to a range of other cations (Rb+, Cs+ and NH4+) also does not change. The asymmetric structural changes that occur during stretch activation therefore allow the channel to respond to changes in membrane tension without a loss of K+ selectivity.


Dynamic role of the tether helix in PIP2-dependent gating of a G protein-gated potassium channel.

The Journal of general physiology (2017)

E Lacin, P Aryal, IW Glaaser, K Bodhinathan, E Tsai, N Marsh, SJ Tucker, MSP Sansom, PA Slesinger

G protein-gated inwardly rectifying potassium (GIRK) channels control neuronal excitability in the brain and are implicated in several different neurological diseases. The anionic phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is an essential cofactor for GIRK channel gating, but the precise mechanism by which PIP2 opens GIRK channels remains poorly understood. Previous structural studies have revealed several highly conserved, positively charged residues in the "tether helix" (C-linker) that interact with the negatively charged PIP2 However, these crystal structures of neuronal GIRK channels in complex with PIP2 provide only snapshots of PIP2's interaction with the channel and thus lack details about the gating transitions triggered by PIP2 binding. Here, our functional studies reveal that one of these conserved basic residues in GIRK2, Lys200 (6'K), supports a complex and dynamic interaction with PIP2 When Lys200 is mutated to an uncharged amino acid, it activates the channel by enhancing the interaction with PIP2 Atomistic molecular dynamic simulations of neuronal GIRK2 with the same 6' substitution reveal an open GIRK2 channel with PIP2 molecules adopting novel positions. This dynamic interaction with PIP2 may explain the intrinsic low open probability of GIRK channels and the mechanism underlying activation by G protein Gβγ subunits and ethanol.


Asymmetric mechanosensitivity in a eukaryotic ion channel.

Proceedings of the National Academy of Sciences of the United States of America 114 (2017) E8343-E8351

MV Clausen, V Jarerattanachat, EP Carpenter, MSP Sansom, SJ Tucker

Living organisms perceive and respond to a diverse range of mechanical stimuli. A variety of mechanosensitive ion channels have evolved to facilitate these responses, but the molecular mechanisms underlying their exquisite sensitivity to different forces within the membrane remains unclear. TREK-2 is a mammalian two-pore domain (K2P) K+ channel important for mechanosensation, and recent studies have shown how increased membrane tension favors a more expanded conformation of the channel within the membrane. These channels respond to a complex range of mechanical stimuli, however, and it is uncertain how differences in tension between the inner and outer leaflets of the membrane contribute to this process. To examine this, we have combined computational approaches with functional studies of oppositely oriented single channels within the same lipid bilayer. Our results reveal how the asymmetric structure of TREK-2 allows it to distinguish a broad profile of forces within the membrane, and illustrate the mechanisms that eukaryotic mechanosensitive ion channels may use to detect and fine-tune their responses to different mechanical stimuli.


The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery.

Accounts of chemical research 50 (2017) 2496-2509

RK O'Reilly, AJ Turberfield, TR Wilks

Precise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible. The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify "successful" structures. The largest libraries made by conventional synthesis are currently of the order of 108 distinct molecules. To put this in context, there are 1013 ways of arranging the 21 proteinogenic amino acids in chains up to 10 units long. Given that we know that a number of these compounds have potent biological activity, it would be highly desirable to be able to search them all to identify leads for new drug molecules. Large libraries of oligonucleotides can be synthesized combinatorially and translated into peptides using systems based on biological replication such as mRNA display, with selected molecules identified by DNA sequencing; but these methods are limited to BBs that are compatible with cellular machinery. In order to search the vast tracts of chemical space beyond nucleic acids and natural peptides, an alternative approach is required. DNA-templated synthesis (DTS) could enable us to meet this challenge. DTS controls chemical product formation by using the specificity of DNA hybridization to bring selected reactants into close proximity, and is capable of the programmed synthesis of many distinct products in the same reaction vessel. By making use of dynamic, programmable DNA processes, it is possible to engineer a system that can translate instructions coded as a sequence of DNA bases into a chemical structure-a process analogous to the action of the ribosome in living organisms but with the potential to create a much more chemically diverse set of products. It is also possible to ensure that each product molecule is tagged with its identifying DNA sequence. Compound libraries synthesized in this way can be exposed to selection against suitable targets, enriching successful molecules. The encoding DNA can then be amplified using the polymerase chain reaction and decoded by DNA sequencing. More importantly, the DNA instruction sequences can be mutated and reused during multiple rounds of amplification, translation, and selection. In other words, DTS could be used as the foundation for a system of synthetic molecular evolution, which could allow us to efficiently search a vast chemical space. This has huge potential to revolutionize materials discovery-imagine being able to evolve molecules for light harvesting, or catalysts for CO2 fixation. The field of DTS has developed to the point where a wide variety of reactions can be performed on a DNA template. Complex architectures and autonomous "DNA robots" have been implemented for the controlled assembly of BBs, and these mechanisms have in turn enabled the one-pot synthesis of large combinatorial libraries. Indeed, DTS libraries are being exploited by pharmaceutical companies and have already found their way into drug lead discovery programs. This Account explores the processes involved in DTS and highlights the challenges that remain in creating a general system for molecular discovery by evolution.


In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria

Nucleic Acids Research 45 (2017) 926-937

A Plochowietz, I Farrell, Z Smilansky, BS Cooperman, AN Kapanidis


Bilayer-Mediated Structural Transitions Control Mechanosensitivity of the TREK-2 K2P Channel.

Structure (London, England : 1993) 25 (2017) 708-718.e2

P Aryal, V Jarerattanachat, MV Clausen, M Schewe, C McClenaghan, L Argent, LJ Conrad, YY Dong, ACW Pike, EP Carpenter, T Baukrowitz, MSP Sansom, SJ Tucker

The mechanosensitive two-pore domain (K2P) K+ channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the "down" to "up" conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer.


A BEST example of channel structure annotation by molecular simulation.

Channels (Austin, Tex.) 11 (2017) 347-353

S Rao, G Klesse, PJ Stansfeld, SJ Tucker, MSP Sansom

An increasing number of ion channel structures are being determined. This generates a need for computational tools to enable functional annotation of channel structures. However, several studies of ion channel and model pores have indicated that the physical dimensions of a pore are not always a reliable indicator of its conductive status. This is due to the unusual behavior of water within nano-confined spaces, resulting in a phenomenon referred to as "hydrophobic gating". We have recently demonstrated how simulating the behavior of water within an ion channel pore can be used to predict its conductive status. In this addendum to our study, we apply this method to compare the recently solved structure of a mutant of the bestrophin chloride channel BEST1 with that of the wild-type channel. Our results support the hypothesis of a hydrophobic gate within the narrow neck of BEST1. This provides further validation that this simulation approach provides the basis for an accurate and computationally efficient tool for the functional annotation of ion channel structures.


Regulation of Two-pore Domain K plus Channels by Natural Effectors and Pharmacological Agents

(2017)

M Schewe, F Schulz, U Mert, H Sun, H Belabed, M Musinszki, T Koehler, M Tegtmeier, M Nazare, EP Carpenter, SJ Tucker, T Baukrowitz


Amphiphilic DNA tiles for controlled insertion and 2D assembly on fluid lipid membranes: the effect on mechanical properties

Nanoscale 9 (2017) 3051-3058

C Dohno, S Makishi, K Nakatani, S Contera


Structural and Functional Response of a Mechanosensitive K2P K+ Channel to Asymmetric Membrane Tension

(2017)

V Jarerattanachat, MV Clausen, P Aryal, EP Carpenter, MSP Sansom, SJ Tucker


Polymodal Gating of the TREK-2 K2P Potassium Channel Involves Structurally Distinct Open States

(2016)

C McClenaghan, M Schewe, T Baukrowitz, SJ Tucker


Nanoscale Metal Particles as Nanocarriers in Targeted Drug Delivery System

Journal of Nanomedicine Research (2016)

N Nurul Akmal Che Lah, M Mahendran Samykano, S Sonia Trigueros


Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences 371 (2016)

D Di Paolo, O Afanzar, JP Armitage, RM Berry

For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.This article is part of the themed issue 'The new bacteriology'.


The Limiting Speed of the Bacterial Flagellar Motor.

Biophysical journal 111 (2016) 557-564

JA Nirody, RM Berry, G Oster

Recent experiments on the bacterial flagellar motor have shown that the structure of this nanomachine, which drives locomotion in a wide range of bacterial species, is more dynamic than previously believed. Specifically, the number of active torque-generating complexes (stators) was shown to vary across applied loads. This finding brings under scrutiny the experimental evidence reporting that limiting (zero-torque) speed is independent of the number of active stators. In this study, we propose that, contrary to previous assumptions, the maximum speed of the motor increases as additional stators are recruited. This result arises from our assumption that stators disengage from the motor for a significant portion of their mechanochemical cycles at low loads. We show that this assumption is consistent with current experimental evidence in chimeric motors, as well as with the requirement that a processive motor driving a large load via an elastic linkage must have a high duty ratio.


Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor.

Nature structural & molecular biology 23 (2016) 197-203

MA Baker, RM Hynson, LA Ganuelas, NS Mohammadi, CW Liew, AA Rey, AP Duff, AE Whitten, CM Jeffries, NJ Delalez, YV Morimoto, D Stock, JP Armitage, AJ Turberfield, K Namba, RM Berry, LK Lee

Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.


A Non-Canonical Voltage Sensor Controls Gating in K2P K+ Channels

(2016)

M Schewe, E Nematian-Ardestani, H Sun, M Musinszki, S Cordeiro, G Bucci, BL de Groot, SJ Tucker, M Rapedius, T Baukrowitz


A Simple low-cost device enables four epi-illumination techniques on standard light microscopes.

Scientific reports 6 (2016) 20729-

RR Ishmukhametov, AN Russell, RJ Wheeler, AL Nord, RM Berry

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

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