Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers Paulina D. Rakowskaa,b,1, Haibo Jiangc,1, Santanu Raya,1, Alice Pynea,d, Baptiste Lamarrea, Matthew Carre, Peter J. Judgef, Jascindra Ravia, Ulla I. M. Gerlingg, Beate Kokschg, Glenn J. Martynah, Bart W. Hoogenboomd,i, Anthony Wattsf, Jason Craina,e, Chris R. M. Grovenorc, and Maxim G. Ryadnova,e,2 aNational Physical Laboratory, Teddington TW11 0LW, United Kingdom; bDepartment of Chemistry, University College London, London WC1H 0AJ, United Kingdom; cDepartment of Materials, University of Oxford, Oxford OX1 3PH, United Kingdom; dLondon Centre for Nanotechnology, University College London, London WC1H 0AH, United Kingdom; eSchool of Physics and Astronomy, University of Edinburgh, Edinburgh EH9 3JZ, United Kingdom; fDepartment of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom; gInstitut für Chemie und Biochemie, Freie Universität Berlin, 14195 Berlin, Germany; hIBM T. J. Watson Research Center, Yorktown Heights, NY 10598; and iDepartment of Physics and Astronomy, University College London, London WC1E 6BT, United Kingdom Edited by Hiroshi Nikaido, University of California, Berkeley, CA, and approved April 16, 2013 (received for review January 2, 2013) Antimicrobial peptides are postulated to disrupt microbial phospho- sufficient to cause the rapid discharge of vital resources (ions, lipid membranes. The prevailing molecular model is based on the ATP) from host cells (19, 20). Cell death in this case is a con- formation of stable or transient pores although the direct observa- sequence, but not an aim. In contrast, AMPs are designed to kill tion of the fundamental processes is lacking. By combining rational microbial cells, not necessarily specifically (21), but rapidly peptide design with topographical (atomic force microscopy) and within the time limits of their proteolytic stability. The behavior chemical (nanoscale secondary ion mass spectrometry) imaging on of other protein toxins is somewhat similar to these two sce- the same samples, we show that pores formed by antimicrobial narios. For example, perforins (22) that activate intrinsic suicide peptides in supported lipid bilayers are not necessarily limited to programs (apoptosis) of various cells, thus mediating cell lysis a particular diameter, nor they are transient, but can expand laterally rather than causing it directly, use different avenues for mem- at the nano-to-micrometer scale to the point of complete membrane brane targeting and can form heterogeneous transmembrane disintegration. The results offer a mechanistic basis for membrane pores (23). Heterogeneity in pore formation for AMPs may de- poration as a generic physicochemical process of cooperative and rive from the fact that, unlike the case of membrane proteins continuous peptide recruitment in the available phospholipid matrix. (24), there are no a priori topological constraints on assembled structures that the peptides must adopt in bilayers. Therefore, innate host defense | de novo protein design | nanometrology | their pore sizes may be governed as much by progressive peptide antibiotics | nanoscopy aggregation as they are by local energetics. Because AMPs are typically cationic, free-energy changes in the edges of pores can tructurally compromised phospholipid membranes can lead be affected by peptide positions and local variations in the di- Sto premature cell death, which is particularly critical for electric medium between peptide molecules, suggesting strong unicellular microorganisms (1, 2). Multicellular organisms take electrostatic repulsion. In this light, poration can be described as the full advantage of such vulnerability by using host defense or a physical phenomenon accommodating peptide diffusion in the antimicrobial peptides (AMPs) (1–6). Although there are >1,000 membrane matrix with no strict predisposition for a particular AMPs known to date (7), only a few have been studied to expose pore size, but with sufficient freedom of movement for lateral the molecular mechanisms of action. The proposed barrel-stave expansion. To address this phenomenon in a sufficient molecular pore (4, 8), torroidal pore (3), and carpet models (9) differ ac- detail, the direct observation of pore architecture and dynamics cording to the ways in which AMPs interact within phospholipid is needed, but thus far has been lacking. bilayers, but all are believed to involve two distinct peptide–lipid One reason for the lack of direct observation is the intrinsic states—an inactive surface-bound S-state and a pore-like insertion complexity of imaging poration in live cells. Membrane binding of I-state (1, 10, 11). However, the link between these two states AMPs is kinetically driven and, in live cells, occurs over timescales and membrane disintegration remains unresolved. of microseconds to minutes (2, 25). Pores need not expand sub- Despite their apparent diversity in structure and modes of ac- stantially because cell death can occur concomitantly as a result of tion, AMPs share common features that make their modulation in membrane leakage and swelling under osmotic pressure and be- model sequences possible. The peptides preferentially target an- cause AMPs can reach and bind to intracellular targets or disrupt ionic microbial surfaces, upon binding to which (S-state) they processes that are crucial to cell viability (protein, DNA, or cell- adopt amphipathic conformations by partitioning polar and hy- wall syntheses) within the same timescales (2, 25). Furthermore, drophobic amino acid side chains (2, 12, 13). Neutron diffraction microbial membranes are curved 3D architectures whose diame- and solid-state nuclear magnetic resonance (ssNMR) spectros- ters do not exceed 2 μm. Pore formation in these membranes can copy suggest that these conformations assemble into perpendicular cause significant variations in membrane tension, which can lead to stacks that close into the pore-like (I-state) structures (5, 11, 14). Here, positive curvature strains (15) and membrane thinning (16) are induced and may precede poration. In lipid vesicles (17, 18) Author contributions: B.W.H., A.W., J.C., C.R.M.G., and M.G.R. designed research; P.D.R., and supported bilayers (16), kinetic studies imply the formation of H.J., S.R., A.P., B.L., M.C., P.J.J., J.R., U.I.M.G., and G.J.M. performed research; U.I.M.G. and B.K. transient pores (6), suggesting that antimicrobial peptides may contributed new reagents/analytic tools; P.D.R., H.J., S.R., A.P., B.L., M.C., P.J.J., J.R., B.K., expand through the monolayers of the lipid bilayers (15–18). G.J.M., B.W.H., A.W., J.C., C.R.M.G., and M.G.R. analyzed data; and M.G.R. wrote the paper. Much research has focused on small and stabilized pores (5, 14, The authors declare no conflict of interest. 15, 17). Growth arrest and uniform sizes of pores conform to the This article is a PNAS Direct Submission. functional and structural rationale of specialized transmembrane Freely available online through the PNAS open access option. proteins but may not be consistent with that of antimicrobial 1P.D.R., H.J., and S.R. contributed equally to this work. peptides. 2To whom correspondence should be addressed. E-mail: [email protected]. α Indeed, bacterial protein toxins such as -hemolysins oligo- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. merize into small 2- to 4-nm pores of defined structure, which is 1073/pnas.1222824110/-/DCSupplemental. 8918–8923 | PNAS | May 28, 2013 | vol. 110 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1222824110 Downloaded by guest on September 26, 2021 “premature” membrane rupture before individual pores can ex- of the same type, next to each other when viewed along the pand substantially. A visible expansion in live cells depends on helical axis (Fig. 1A and Fig. S1). This order ensures the segre- these interrelated factors, which makes its direct observation gation of hydrophobic and polar residues onto distinct regions or problematic. faces, giving rise to an amphipathic helix. The hydrophobic face In contrast, longer time and length scale studies are accessible was kept short at the 1:1.5 ratio of hydrophobic (leucine) to in supported lipid bilayers (SLBs) (26). SLBs provide ideal ex- cationic (lysine) residues to avoid hemolytic activities common for perimental models for fluid-phase membranes and can be im- venom peptides that have broader hydrophobic clusters (4, 33). aged by atomic force microscopy (AFM) (27, 28). Combining To support the ratio, small alanines and neutral glutamines, AFM with high-resolution secondary-ion mass spectrometry which do not contribute to membrane binding, were alternately (SIMS), which has been shown to provide compositional chem- arranged in the polar face as a neutral cluster opposite to the ical imaging of small lipid domains with lateral resolution of hydrophobic face (SI Text). <100 nm (29), permits the detailed visualization of poration Peptide folding in solution was probed using zwitterionic uni- in SLBs. lamellar vesicles (ZUVs) and anionic unilamellar vesicles (AUVs) To mitigate the complexities of direct live cell studies, we in- mimicking mammalian and microbial membranes, respectively (33, troduce and explore here a model system designed to expose the 34). Dilauroylphosphatidylcholine (DLPC) was used to assemble fundamental physicochemical processes relevant in the peptide– ZUVs whereas its mixtures with dilaurylphosphatidylglycerol bilayer interactions. A model antimicrobial peptide, which com- (DLPG) at 3:1 ratios were used to assemble AUVs (30, 33). These bines main features of helical AMPs, was designed to integrate lipid compositions yield fluid-phase membranes at room and into SLBs that were then used as substrates for detailed nanoscale physiological temperatures (27, 30, 31). Circular dichroism (CD) imaging and analysis by AFM and high resolution SIMS. spectroscopy revealed that amhelin did not fold in aqueous buffers or in the presence of ZUVs at micromolar concentrations. In Results contrast, an appreciable helical signal was recorded for the peptide To enable pore formation, a de novo amino acid sequence, in AUV samples (Fig.