RESUMO
Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.
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How alpha-helical membrane proteins fold correctly in the highly hydrophobic membrane interior is not well understood. Their folding is known to be highly influenced by the lipids within the surrounding bilayer, but the majority of folding studies have focused on detergent-solubilized protein rather than protein in a lipid environment. There are different ways to study folding in lipid bilayers, and each method has its own advantages and disadvantages. This review will discuss folding methods which can be used to study alpha-helical membrane proteins in bicelles, liposomes, nanodiscs or native membranes. These folding methods include in vitro folding methods in liposomes such as denaturant unfolding studies, and single-molecule force spectroscopy studies in bicelles, liposomes and native membranes. This review will also discuss recent advances in co-translational folding studies, which use cell-free expression with liposomes or nanodiscs or are performed in vivo with native membranes.
Assuntos
Lipossomos , Proteínas de Membrana , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Conformação Proteica em alfa-Hélice , Dobramento de ProteínaRESUMO
Membrane proteins need to fold with precision in order to function correctly, with misfolding potentially leading to disease. The proteins reside within a hydrophobic lipid membrane and must insert into the membrane and fold correctly, generally whilst they are being translated by the ribosome. Favourable and unfavourable free energy contributions are present throughout each stage of insertion and folding. The unfavourable energy cost of transferring peptide bonds into the hydrophobic membrane interior is compensated for by the favourable hydrophobic effect of partitioning a hydrophobic transmembrane alpha-helix into the membrane. Native membranes are composed of many different types of lipids, but how these different lipids influence folding and the associated free energies is not well understood. Altering the lipids in the bilayer is known to affect the probability of transmembrane helix insertion into the membrane, and lipids also affect protein stability and can promote successful folding. This review will summarise the free energy contributions associated with insertion and folding of alpha helical membrane proteins, as well as how lipids can make these processes more or less favourable. We will also discuss the implications of this work for the free energy landscape during the co-translational folding of alpha helical membrane proteins.
Assuntos
Proteínas de Membrana , Dobramento de Proteína , Bicamadas Lipídicas/química , Lipídeos/química , Proteínas de Membrana/metabolismo , Conformação Proteica em alfa-Hélice , Ribossomos/metabolismoRESUMO
The majority of alpha helical membrane proteins fold co-translationally during their synthesis on the ribosome. In contrast, most mechanistic folding studies address refolding of full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation of membrane proteins is emerging as a useful tool to address folding during translation by a ribosome. We summarise the benefits of this approach and show how it can be successfully extended to a membrane protein with a complex topology. The bacterial leucine transporter, LeuT can be synthesised and inserted into lipid membranes using a variety of in vitro transcription translation systems. Unlike major facilitator superfamily transporters, where changes in lipids can optimise the amount of correctly inserted protein, LeuT insertion yields are much less dependent on the lipid composition. The presence of a bacterial translocon either in native membrane extracts or in reconstituted membranes also has little influence on the yield of LeuT incorporated into the lipid membrane, except at high reconstitution concentrations. LeuT is considered a paradigm for neurotransmitter transporters and possesses a knotted structure that is characteristic of this transporter family. This work provides a method in which to probe the formation of a protein as the polypeptide chain is being synthesised on a ribosome and inserting into lipids. We show that in comparison with the simpler major facilitator transporter structures, LeuT inserts less efficiently into membranes when synthesised cell-free, suggesting that more of the protein aggregates, likely as a result of the challenging formation of the knotted topology in the membrane.
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In order to comprehend the molecular basis of transmembrane protein biogenesis, methods are required that are capable of investigating the co-translational folding of these hydrophobic proteins. Equally, in artificial cell studies, controllable methods are desirable for in situ synthesis of membrane proteins that then direct reactions in the synthetic cell membrane. Here we describe a method that exploits cell-free expression systems and tunable membrane mimetics to facilitate co-translational studies. Alteration of the lipid bilayer composition improves the efficiency of the folding system. The approach also enables membrane transport proteins to be made and inserted into artificial cell platforms such as droplet interface bilayers. Importantly, this gives a new facet to the droplet networks by enabling specific transport of molecules across the synthetic bilayer against a concentration gradient. This method also includes a protocol to pause and restart translation of membrane proteins at specified positions during their co-translational folding. This stop-start strategy provides an avenue to investigate whether the proteins fold in sequence order, or if the correct fold of N-terminal regions is reliant on the synthesis of downstream residues.
Assuntos
Bicamadas Lipídicas , Dobramento de Proteína , Sistema Livre de Células/metabolismo , Bicamadas Lipídicas/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend, features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC/DOPE/DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.
Assuntos
Bicamadas Lipídicas , Fosfatidilcolinas , Proteínas de Membrana Transportadoras , Lipossomas UnilamelaresRESUMO
Droplet interface bilayers (DIBs) are an emerging tool within synthetic biology that aims to recreate biological processes in artificial cells. A critical component for the utility of these bilayers is controlled flow between compartments and, notably, uphill transport against a substrate concentration gradient. A versatile method to achieve the desired flow is to exploit the specificity of membrane proteins that regulate the movement of ions and transport of specific metabolic compounds. Methods have been in existence for some time to synthesize proteins within a droplet as well as incorporate membrane proteins into DIBS; however, there have been few reports combining synthesis and DIB incorporation for membrane transporters that demonstrate specific, uphill transport. This chapter presents two methods for the incorporation of a membrane transporter into a simple two-droplet DIB system, with the downhill and uphill transport reaction readily monitored by fluorescence microscopy.
Assuntos
Bicamadas Lipídicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Íons/metabolismo , Microscopia de Fluorescência/métodos , Movimento/fisiologiaRESUMO
Droplet microcompartments linked by lipid bilayers show great promise in the construction of synthetic minimal tissues. Central to controlling the flow of information in these systems are membrane proteins, which can gate in response to specific stimuli in order to control the molecular flux between membrane separated compartments. This has been demonstrated with droplet interface bilayers (DIBs) using several different membrane proteins combined with electrical, mechanical, and/or chemical activators. Here we report the activation of the bacterial mechanosensitive channel of large conductance (MscL) in a dioleoylphosphatidylcholine:dioleoylphosphatidylglycerol DIB by controlling membrane asymmetry. We show using electrical measurements that the incorporation of lysophosphatidylcholine (LPC) into one of the bilayer leaflets triggers MscL gating in a concentration-dependent manner, with partial and full activation observed at 10 and 15 mol% LPC respectively. Our findings could inspire the design of new minimal tissues where flux pathways are dynamically defined by lipid composition.
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Structure and function analysis of human membrane proteins in lipid bilayer environments is acutely lacking despite the fundame1ntal cellular importance of these proteins and their dominance of drug targets. An underlying reason is that detailed study usually requires a potentially destabilising detergent purification of the proteins from their host membranes prior to subsequent reconstitution in a membrane mimic; a situation that is exacerbated for human membrane proteins due to the inherent difficulties in overexpressing suitable quantities of the proteins. We advance the promising styrene maleic acid polymer (SMA) extraction approach to introduce a detergent-free method of obtaining stable, functional human membrane transporters in bilayer nanodiscs directly from yeast cells. We purify the human serotonin transporter (hSERT) following overexpression in Pichia pastoris using diisobutylene maleic acid (DIBMA) as a superior method to traditional detergents or the more established styrene maleic acid polymer. hSERT plays a pivotal role in neurotransmitter regulation being responsible for the transport of the neurotransmitter 5-hydroxytryptamine (5-HT or serotonin). It is representative of the neurotransmitter sodium symporter (NSS) family, whose importance is underscored by the numerous diseases attributed to their malfunction. We gain insight into hSERT activity through an in vitro transport assay and find that DIBMA extraction improves the thermostability and activity of hSERT over the conventional detergent method.
Assuntos
Alcenos/química , Maleatos/química , Polímeros/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Humanos , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/isolamento & purificação , TemperaturaRESUMO
OBJECTIVE: To systematically examine the effect of dehydration on health outcomes, identify associated financial costs and consider impacts on cognitive performance in older adults. DESIGN: A systematic review of English-language articles via OVID using MEDLINE, PsychINFO, EMBASE, and others, to March 2018. Included studies examined the relationship between hydration status and health, care costs or cognitive outcome. SETTING: Cross sectional and cohort data from studies reporting on dehydration in older adults. PARTICIPANTS: Adults aged 60 years and older. MEASUREMENTS: Independent quality ratings were assessed for all extracted articles. RESULTS: Of 1684 articles screened, 18 papers (N = 33,707) met inclusion criteria. Participants were recruited from hospital settings, medical long-term care centres and the community dwelling population. Data were synthesised using a narrative summary. Mortality rates were higher in dehydrated patients. Furthermore, health outcomes, including frailty, bradyarrhythmia, transient ischemic attacks, oral health and surgery recovery are linked to and worsened by dehydration. Length of hospital stay, either as a principal or secondary diagnosis, is greater in those with dehydration, compared to those who are euhydrated. Finally, neurocognitive functioning may be impacted by dehydration. There are issues with study design, inconsistency in hydration status measurement and different measures used for outcome assessment. CONCLUSION: Dehydration in older people is associated with increased mortality, poorer course of illness and increased costs for health services. In addition, there is some, but sparse evidence that dehydration in older people is linked to poorer cognitive performance. Intervention studies should test strategies for reducing dehydration in older adults.
Assuntos
Desidratação , Custos de Cuidados de Saúde , Idoso , Cognição , Estudos Transversais , Humanos , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de SaúdeRESUMO
Proton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.
Assuntos
Proteínas de Escherichia coli/química , Glucose/química , Prótons , Simportadores/química , Xilose/química , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Medição da Troca de Deutério , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glucose/metabolismo , Espectrometria de Massa com Troca Hidrogênio-Deutério , Cinética , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Simportadores/antagonistas & inibidores , Simportadores/genética , Simportadores/metabolismo , Termodinâmica , Xilose/metabolismoRESUMO
Resistance-nodulation-division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics.
Assuntos
Ciprofloxacina/farmacologia , Dipeptídeos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Quinases/química , Antibacterianos/química , Antibacterianos/farmacologia , Sítios de Ligação/genética , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Ciprofloxacina/química , Dicroísmo Circular , Deutério/química , Dipeptídeos/química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligantes , Espectrometria de Massas/métodos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Proteínas Quinases/genética , Proteínas Quinases/metabolismoRESUMO
Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo-generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene-maleic acid co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milieu.
Assuntos
Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Alcenos/química , Sequência de Aminoácidos , Maleatos/química , Micelas , Nanopartículas/química , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas/química , Canais de Translocação SEC/metabolismoRESUMO
Most helical membrane proteins fold co-translationally during unidirectional polypeptide elongation by the ribosome. Studies thus far, however, have largely focussed on refolding full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation offers opportunities to remedy this deficit in folding studies and has previously been used for membrane proteins. We exploit this cell-free approach to develop tools to probe co-translational folding. We show that two transporters from the ubiquitous Major Facilitator Superfamily can successfully insert into a synthetic bilayer without the need for translocon insertase apparatus that is essential in vivo. We also assess the cooperativity of domain insertion, by expressing the individual transporter domains cell-free. Furthermore, we manipulate the cell-free reaction to pause and re-start protein synthesis at specific points in the protein sequence. We find that full-length protein can still be made when stalling after the first N terminal helix has inserted into the bilayer. However, stalling after the first three helices have exited the ribosome cannot be successfully recovered. These three helices cannot insert stably when ribosome-bound during co-translational folding, as they require insertion of downstream helices.
Assuntos
Proteínas de Membrana Transportadoras/química , Sistema Livre de Células , Bases de Dados de Proteínas , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lipossomos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutagênese Sítio-Dirigida , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Simportadores/química , Simportadores/genética , Simportadores/metabolismoRESUMO
Membrane function is fundamental to life. Each species explores membrane lipid diversity within a genetically predefined range of possibilities. How membrane lipid composition in turn defines the functional space available for evolution of membrane-centered processes remains largely unknown. We address this fundamental question using related fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus. We show that, unlike S. pombe that generates membranes where both glycerophospholipid acyl tails are predominantly 16-18 carbons long, S. japonicus synthesizes unusual "asymmetrical" glycerophospholipids where the tails differ in length by 6-8 carbons. This results in stiffer bilayers with distinct lipid packing properties. Retroengineered S. pombe synthesizing the S.-japonicus-type phospholipids exhibits unfolded protein response and downregulates secretion. Importantly, our protein sequence comparisons and domain swap experiments support the hypothesis that transmembrane helices co-evolve with membranes, suggesting that, on the evolutionary scale, changes in membrane lipid composition may necessitate extensive adaptation of the membrane-associated proteome.
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Evolução Molecular , Lipídeos de Membrana/química , Proteínas de Membrana/química , Membrana Nuclear/química , Schizosaccharomyces/química , Especificidade da EspécieRESUMO
Droplet interface bilayers (DIBs) are model membranes formed between lipid monolayer-encased water droplets in oil. Compared to conventional methods, one of the most unique properties of DIBs is that they can be connected together to generate multi-layered 'tissue-like' networks, however introducing communication pathways between these compartments typically relies on water-soluble pores that are unable to gate. Here, we show that network connectivity can instead be achieved using a water-insoluble membrane protein by successfully reconstituting a chemically activatable mutant of the mechanosensitive channel MscL into a network of DIBs. Moreover, we also show how the small molecule activator can diffuse through an open channel and across the neighbouring droplet to activate MscL present in an adjacent bilayer. This demonstration of membrane protein mediated bilayer communication could prove key toward developing the next generation of responsive bilayer networks capable of defining information flow inside a minimal tissue.
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Membrane protein folding studies lag behind those of water-soluble proteins due to immense difficulties of experimental study, resulting from the need to provide a hydrophobic lipid-bilayer environment when investigated in vitro. A sound understanding of folding mechanisms is important for membrane proteins as they contribute to a third of the proteome and are frequently associated with disease when mutated and/or misfolded. Membrane proteins largely consist of α-helical, hydrophobic transmembrane domains, which insert into the membrane, often using the SecYEG/Sec61 translocase system. This mini-review highlights recent advances in techniques that can further our understanding of co-translational folding and notably, the structure and insertion of nascent chains as they emerge from translating ribosomes. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.
Assuntos
Proteínas de Membrana/química , Dobramento de Proteína , Sistemas de Translocação de Proteínas/fisiologia , Animais , Humanos , Ribossomos/fisiologia , Canais de Translocação SEC/metabolismoRESUMO
To date, reconstitution of one of the fundamental methods of cell communication, the signaling pathway, has been unaddressed in the bottom-up construction of artificial cells (ACs). Such developments are needed to increase the functionality and biomimicry of ACs, accelerating their translation and application in biotechnology. Here, we report the construction of a de novo synthetic signaling pathway in microscale nested vesicles. Vesicle-cell models respond to external calcium signals through activation of an intracellular interaction between phospholipase A2 and a mechanosensitive channel present in the internal membranes, triggering content mixing between compartments and controlling cell fluorescence. Emulsion-based approaches to AC construction are therefore shown to be ideal for the quick design and testing of new signaling networks and can readily include synthetic molecules difficult to introduce to biological cells. This work represents a foundation for the engineering of multicompartment-spanning designer pathways that can be utilized to control downstream events inside an AC, leading to the assembly of micromachines capable of sensing and responding to changes in their local environment.
Assuntos
Células Artificiais , Compartimento Celular , Mecanotransdução Celular , Cálcio/metabolismo , Comunicação Celular/efeitos dos fármacos , Compartimento Celular/efeitos dos fármacos , Quelantes/farmacologia , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular/efeitos dos fármacos , Fosfolipases A2/metabolismoRESUMO
OBJECTIVES: This study explored the potential for e-cigarette advertisements to (1) enhance attitudes towards cigarettes and/or (2) reduce barriers to e-cigarettes uptake. The study tested whether exposure to an online electronic cigarette advertisement changed attitudes towards cigarettes and e-cigarettes in smokers, non-smokers, e-cigarette users and dual users (smokers who also use e-cigarettes). DESIGN: Cross-sectional study SETTING: Online survey PARTICIPANTS: Adults (n=964) aged 18 to 65 years old (M=36 years, SD=11.6) from the UK and USA. Participants were grouped into current non-smokers, e-cigarette users, dual users and smokers. INTERVENTIONS: Participants viewed 1 of 15 randomly assigned online e-cigarette advertisements. PRIMARY MEASURES: Three single seven-point Likert scales measuring health, desirability, social acceptability were completed pre and post advertisement exposure. RESULTS: Post exposure all smoking groups showed a decrease or no change in how socially acceptable or desirable they rated cigarettes. Paradoxically, dual users rated cigarettes as being significantly healthier after viewing the advertisement (p=0.01) while all other smoking group ratings remained the same. There was an increase or no change in how all smoking groups perceived the healthiness and desirability of e-cigarettes CONCLUSIONS: We observed no evidence that exposure to an e-cigarette advertisement renormalises or encourages smoking in smokers, non-smokers or e-cigarette users. However, there is some indication that viewing an e-cigarette advertisement may increase duals users' perceptions of the health of smoking.