Transmembrane peptide effects on bacterial membrane integrity and organization

  • Chloe J Mitchell
    Program in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4, Ontario, Canada

    Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada
    Search for articles by this author
  • Tyler S. Johnson
    Program in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4, Ontario, Canada

    Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada
    Search for articles by this author
  • Charles M. Deber
    Corresponding author
    Program in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4, Ontario, Canada

    Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada
    Search for articles by this author
Published:August 02, 2022DOI:


      As the bacterial multidrug resistance crisis continues, membrane-active antimicrobial peptides are being explored as an alternate treatment to conventional antibiotics. In contrast to antimicrobial peptides, which function by a nonspecific membrane disruption mechanism, here we describe a series of transmembrane (TM) peptides that are designed to act as drug efflux inhibitors by aligning with and out-competing a conserved TM4-TM4 homodimerization motif within bacterial small multidrug resistance proteins. The peptides contain two terminal tags: a C-terminal lysine tag to direct the peptides toward the negatively charged bacterial membrane, and an uncharged N-terminal sarcosine (N-methyl-glycine) tag to promote membrane insertion. While effective at inhibiting efflux activity, ostensibly through their designed mechanism of action, the impact of the peptides on the bacterial inner membrane remains undetermined. To evaluate the extant peptide-membrane interactions, we performed a series of biophysical measurements. Circular dichroism spectroscopy and Trp fluorescence showed that the peptides insert into the membrane generally in helical form. Interestingly, differential scanning calorimetry of the peptides added to bacterial-like membranes (POPE:POPG 3:1) revealed the peptides’ ability to demix the POPE and POPG lipids, creating two pools, one of which is likely a peptide-POPG conglomerate, and the other a POPE-rich component where the native POPG content has been depleted. However, dye leakage assays confirmed that these events occur without causing significant membrane disruption both in vitro and in vivo, indicating that the peptides can target the small multidrug resistance TM4-TM4 motif without nonspecific membrane disruption. In related studies, DiOC2(3) fluorescence indicated moderate peptide-mediated reduction of the proton motive force for all peptides, including control peptides that did not display inhibitory activity. The overall findings suggest that peptides designed with suitable tags, sequence hydrophobicity, and charge distribution can be directed more generally to impact proteins whose function involves membrane-embedded protein-protein interactions.
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Biophysical Journal
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Avci F.G.
        • Akbulut B.S.
        • Ozkirimli E.
        Membrane active peptides and their biophysical characterization.
        Biomolecules. 2018; 8: 77
        • World Heath Organization
        Antibacterial Agents in Clinical Development. An Analysis of the Antibacterial Clinical Development Pipeline, Including Tuberculosis.
        • Centers for Disease Control and Prevention
        Biggest Threats and Data: 2019 AR Threats Report. 2019.
        • Albrecht C.
        • Appert-Collin A.
        • Bennasroune A.
        • et al.
        Transmembrane peptides as inhibitors of protein-protein interactions: an efficient strategy to target cancer cells?.
        Front. Oncol. 2020; 10: 519
        • Bennasroune A.
        • Fickova M.
        • Hubert P.
        • et al.
        Transmembrane peptides as inhibitors of ErbB receptor signaling.
        Mol. Biol. Cell. 2004; 15: 3464-3474
        • Arpel A.
        • Sawma P.
        • Bagnard D.
        • et al.
        Transmembrane domain targeting peptide antagonizing ErbB2/Neu inhibits breast tumor growth and metastasis.
        Cell Rep. 2014; 8: 1714-1721
        • Gamper C.
        • Spenlé C.
        • Heinlein M.
        • et al.
        Functionalized tobacco mosaic virus coat protein monomers and oligomers as nanocarriers for anti-cancer peptides.
        Cancers. 2019; 11: 1609
        • Harikumar K.G.
        • Pinon D.I.
        • Miller L.J.
        Transmembrane segment IV contributes a functionally important interface for oligomerization of the class II G protein-coupled secretin receptor.
        J. Biol. Chem. 2007; 282: 30363-30372
        • Borroto-Escuela D.O.
        • Rodriguez D.
        • Carlsson J.
        • et al.
        Mapping the interface of a GPCR Dimer: a structural model of the A2A Adenosine and D2 dopamine receptor heteromer.
        Front. Pharmacol. 2018; 9: 1-16
        • Gallo M.
        • Navarro G.
        • Andreu D.
        • et al.
        A2A receptor homodimer-disrupting sequence efficiently delivered by a protease-resistant, cyclic cpp vector.
        Int. J. Mol. Sci. 2019; 20: 4937
        • Borroto-Escuela D.O.
        • Wydra K.
        • Fuxe K.
        • et al.
        Disruption of A2AR-D2R heteroreceptor complexes after A2AR transmembrane 5 peptide administration enhances cocaine self-administration in rats.
        Mol. Neurobiol. 2018; 55: 7038-7048
        • Bellmann-Sickert K.
        • Stone T.A.
        • Deber C.M.
        • et al.
        Efflux by small multidrug resistance proteins is inhibited by membrane-interactive helix-stapled peptides.
        J. Biol. Chem. 2015; 290: 1752-1759
        • Poulsen B.E.
        • Deber C.M.
        Drug efflux by a small multidrug resistance protein is inhibited by a transmembrane peptide.
        Antimicrob. Agents Chemother. 2012; 56: 3911-3916
        • Jesin J.A.
        • Stone T.A.
        • Deber C.M.
        • et al.
        Peptide-based approach to inhibition of the multidrug resistance efflux pump AcrB.
        Biochemistry. 2020; 59: 3973-3981
        • Mitchell C.J
        • Stone T.A.
        • Deber C.M.
        Peptide-based efflux pump inhibitors of the small multidrug resistance protein from Pseudomonas aeruginosa.
        Antimicrob. Agents Chemother. 2019; 63 (e00730-19-19)
        • Bay D.C.
        • Rommens K.L.
        • Turner R.J.
        Small multidrug resistance proteins: a multidrug transporter family that continues to grow.
        Biochim. Biophys. Acta. 2008; 1778: 1814-1838
        • Bay D.C.
        • Turner R.J.
        Diversity and evolution of the small multidrug resistance protein family.
        BMC Evol. Biol. 2009; 9: 140
        • Robinson A.E.
        • Thomas N.E.
        • Henzler-Wildman K.A.
        • et al.
        New free-exchange model of EmrE transport.
        Proc. Natl. Acad. Sci. USA. 2017; 114: E10083-E10091
        • Hussey G.A.
        • Thomas N.E.
        • Henzler-Wildman K.A.
        Highly coupled transport can be achieved in free-exchange transport models.
        J. Gen. Physiol. 2020; 152: e201912437
        • Chen Y.-J.
        • Pornillos O.
        • Chang G.
        • et al.
        X-ray structure of EmrE supports dual topology model.
        Proc. Natl. Acad. Sci. USA. 2007; 104: 18999-19004
        • Poulsen B.E.
        • Rath A.
        • Deber C.M.
        The assembly motif of a bacterial small multi drug resistance protein.
        J. Biol. Chem. 2009; 284: 9870-9875
        • Glukhov E.
        • Burrows L.L.
        • Deber C.M.
        Membrane interactions of designed cationic antimicrobial peptides: the two thresholds.
        Biopolymers. 2008; 89: 360-371
        • Melnyk R.A.
        • Partridge A.W.
        • Deber C.M.
        • et al.
        Polar residue tagging of transmembrane peptides.
        Biopolymers. 2003; 71: 675-685
        • Tang Y.C.
        • Deber C.M.
        Hydrophobicity and helicity of membrane-interactive peptides containing peptoid residues.
        Biopolymers. 2002; 65: 254-262
        • Shcherbakov A.A.
        • Hisao G.
        • Hong M.
        • et al.
        Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers.
        Nat. Commun. 2021; 12: 172
        • Snider C.
        • Jayasinghe S.
        • White S.H.
        • et al.
        MPEx: a tool for exploring membrane proteins.
        Protein Sci. 2009; 18: 2624-2628
        • White S.H.
        • Wimley W.C.
        Membrane protein folding and stability: physical Principles.
        Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365
        • Chen Y.H.
        • yang J.T.
        • Chau K.H.
        Determination of the Helix and beta form of proteins in aqueous solution by circular dichroism.
        Biochemistry. 1974; 13: 3350-3359
        • Lacroix E.
        • Viguera A.R.
        • Serrano L.
        Elucidating the folding problem of α-helices: local motifs, long-range electrostatics, lonic-strength dependence and prediction of NMR parameters.
        J. Mol. Biol. 1998; 284: 173-191
        • Stone T.A.
        • Schiller N.
        • Deber C.M.
        • et al.
        Hydrophobic clusters raise the threshold hydrophilicity for insertion of transmembrane sequences in vivo.
        Biochemistry. 2016; 55: 5772-5779
        • Stiefel P.
        • Schmidt-Emrich S.
        • Ren Q.
        • et al.
        Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide.
        BMC Microbiol. 2015; 15: 36
        • Bay D.C.
        • Turner R.J.
        Diversity and evolution of the small multidrug resistance protein family.
        BMC Evol. Biol. 2009; 9: 140
        • Almeida P.F.
        • Pokorny A.
        Mechanisms of antimicrobial, cytolytic, and cell-penetrating peptides: from kinetics to thermodynamics.
        Biochemistry. 2009; 48: 8083-8093
        • Koehbach J.
        • Craik D.J.
        The vast structural diversity of antimicrobial peptides.
        Trends Pharmacol. Sci. 2019; 40: 517-528
        • McAuley S.
        • Huynh A.
        • Czarny T.L.
        • Brown E.D.
        • Nodwell J.R.
        Membrane activity profiling of small molecule: B. subtilis growth inhibitors utilizing novel duel-dye fluorescence assay.
        Medchemcomm. 2018; 9: 554-561
        • Huan Y.
        • Kong Q.
        • Yi H.
        • et al.
        Antimicrobial peptides: classification, design, application and research progress in multiple fields.
        Front. Microbiol. 2020; 11: 582779
        • Epand R.M.
        • Walker C.
        • Magarvey N.A.
        • et al.
        Molecular mechanisms of membrane targeting antibiotics.
        Biochim. Biophys. Acta. 2016; 1858: 980-987
        • Brogden K.A.
        Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?.
        Nat. Rev. Microbiol. 2005; 3: 238-250
        • Kabelka I.
        • Vácha R.
        Advances in molecular understanding of α-helical membrane-active peptides.
        Acc. Chem. Res. 2021; 54: 2196-2204
        • Sani M.A.
        • Separovic F.
        How membrane-active peptides get into lipid membranes.
        Acc. Chem. Res. 2016; 49: 1130-1138
        • Kabelka I.
        • Vácha R.
        Optimal hydrophobicity and reorientation of amphiphilic peptides translocating through membrane.
        Biophys. J. 2018; 115: 1045-1054
        • Yin L.M.
        • Edwards M.A.
        • Deber C.M.
        • et al.
        Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions.
        J. Biol. Chem. 2012; 287: 7738-7745
        • Garidel P.
        • Blume A.
        Miscibility of phosphatidylethanolamine-phosphatidylglycerol mixtures as a function of pH and acyl chain length.
        Eur. Biophys. J. 2000; 28: 629-638
        • Mosior M.
        • McLaughlin S.
        Binding of basic peptides to acidic lipids in membranes: effects of inserting alanine(s) between the basic residues.
        Biochemistry. 1992; 31: 1767-1773
        • Garidel P.
        • Blume A.
        Interaction of alkaline earth cations with the negatively charged phospholipid 1, 2-Dimyristoyl-sn-glycero-3-phosphoglycerol: a differential scanning and isothermal titration calorimetric study.
        Langmuir. 1999; 15: 5526-5534
        • Hoernke M.
        • Schwieger C.
        • Blume A.
        • et al.
        Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: complex interplay of electrostatic and hydrophobic interactions.
        Biochim. Biophys. Acta. 2012; 1818: 1663-1672
        • Arouri A.
        • Dathe M.
        • Blume A.
        Peptide induced demixing in PG/PE lipid mixtures: a mechanism for the specificity of antimicrobial peptides towards bacterial membranes?.
        Biochim. Biophys. Acta. 2009; 1788: 650-659
        • Mason A.J.
        • Martinez A.
        • Bechinger B.
        • et al.
        The antibiotic and DNA-transfecting peptide LAH4 selectively associates with, and disorders, anionic lipids in mixed membranes.
        Faseb. J. 2006; 20: 320-322
        • Munhoz V.H.O.
        • Ferreira C.S.
        • Verly R.M.
        • et al.
        Epimers L- and D-Phenylseptin: how the relative stereochemistry affects the peptide-membrane interactions.
        Biochim. Biophys. Acta Biomembr. 2021; 1863: 183708
        • Joanne P.
        • Galanth C.
        • Alves I.D.
        • et al.
        Lipid reorganization induced by membrane-active peptides probed using differential scanning calorimetry.
        Biochim. Biophys. Acta. 2009; 1788: 1772-1781
        • Scheinpflug K.
        • Wenzel M.
        • Strahl H.
        • et al.
        Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis.
        Sci. Rep. 2017; 7: 44332
        • Mörs K.
        • Hellmich U.A.
        • Glaubitz C.
        • et al.
        A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein TBsmr.
        Biochim. Biophys. Acta Biomembr. 2013; 1828: 561-567
        • Curnow P.
        • Lorch M.
        • Booth P.J.
        • et al.
        The reconstitution and activity of the small multidrug transporter EmrE is modulated by non-bilayer lipid composition.
        J. Mol. Biol. 2004; 343: 213-222
        • Charalambous K.
        • Miller D.
        • Booth P.J.
        • et al.
        Lipid bilayer composition influences small multidrug transporters.
        BMC Biochem. 2008; 9: 31
        • Aisenbrey C.
        • Salnikov E.S.
        • Bechinger B.
        Solid-State NMR investigations of the MHC II transmembrane domains: topological equilibria and lipid interactions.
        J. Membr. Biol. 2019; 252: 371-384
        • Salnikov E.S.
        • Aisenbrey C.
        • Bechinger B.
        • et al.
        Structure, topology, and dynamics of membrane-inserted polypeptides and lipids by solid-state NMR spectroscopy: investigations of the transmembrane domains of the DQ beta-1 subunit of the MHC II receptor and of the COP I protein p24.
        Front. Mol. Biosci. 2019; 6: 1-14
        • Li J.
        • Koh J.J.
        • Liu S.
        • Beuerman R.W.
        • et al.
        Membrane active antimicrobial peptides: translating mechanistic insights to design.
        Front. Neurosci. 2017; 11: 73