Amino Acids

, Volume 50, Issue 5, pp 557–568 | Cite as

Understanding the antimicrobial properties/activity of an 11-residue Lys homopeptide by alanine and proline scan

  • P. Carvajal-Rondanelli
  • M. Aróstica
  • C. A. Álvarez
  • C. Ojeda
  • F. Albericio
  • L. F. Aguilar
  • S. H. Marshall
  • F. Guzmán
Original Article


Previous work demonstrated that lysine homopeptides adopt a polyproline II (PPII) structure. Lysine homopeptides with odd number of residues, especially with 11 residues (K11), were capable of inhibiting the growth of a broader spectrum of bacteria than those with an even number. Confocal studies also determined that K11 was able to localize exclusively in the bacterial membrane, leading to cell death. In this work, the mechanism of action of this peptide was further analyzed focused on examining the structural changes in bacterial membrane induced by K11, and in K11 itself when interacting with bacterial membrane lipids. Moreover, alanine and proline scans were performed for K11 to identify relevant positions in structure conformation and antibacterial activity. To do so, circular dichroism spectroscopy (CD) was conducted in saline phosphate buffer (PBS) and in lipidic vesicles, using large unilamellar vesicles (LUV), composed of 2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or bacterial membrane lipid. Antimicrobial activity of K11 and their analogs was evaluated in Gram-positive and Gram-negative bacterial strains. The scanning electron microscopy (SEM) micrographs of Staphylococcus aureus ATCC 25923 exposed to the Lys homopeptide at MIC concentration showed blisters and bubbles formed on the bacterial surface, suggesting that K11 exerts its action by destabilizing the bacterial membrane. CD analysis revealed a remarkably enhanced PPII structure of K11 when replacing some of its central residues by proline in PBS. However, when such peptide analogs were confronted with either DMPG-LUV or membrane lipid extract-LUV, the tendency to form PPII structure was severely weakened. On the contrary, K11 peptide showed a remarkably enhanced PPII structure in the presence of DMPG-LUV. Antibacterial tests revealed that K11 was able to inhibit all tested bacteria with an MIC value of 5 µM, while proline and alanine analogs have a reduced activity on Listeria monocytogenes. Besides, the activity against Vibrio parahaemolyticus was affected in most of the alanine-substituted analogs. However, lysine substitutions by alanine or proline at position 7 did not alter the activity against all tested bacterial strains, suggesting that this position can be screened to find a substitute amino acid yielding a peptide with increased antibacterial activity. These results also indicate that the PPII secondary structure of K11 is stabilized by the interaction of the peptide with negatively charged phospholipids in the bacterial membrane, though not being the sole determinant for its antimicrobial activity.


Lysine homopeptide-antimicrobial activity-Ala Pro scanning-membrane rupture 



This work was supported by the Chilean Fondo Nacional de Desarrollo Científico y Tecnológico (Fondecyt), Grant 1140926. Doctoral fellowship Pontificia Universidad Católica de Valparaíso. 

Compliance with ethical standards

Fanny Guzmán, on behalf of all authors, declares that the manuscript has not been submitted to any other journal.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

726_2018_2542_MOESM1_ESM.docx (35 kb)
Supplementary material 1 (DOCX 34 kb)


  1. Almaaytah A, Tarazi S, Abu-Alhaijaa A et al (2014) Enhanced antimicrobial activity of AamAP1-lysine, a novel synthetic peptide analog derived from the scorpion venom peptide AamAP1. Pharmaceuticals (Basel) 7:502–516. CrossRefGoogle Scholar
  2. Azkargorta M, Soria J, Ojeda C et al (2015) Human basal tear peptidome characterization by CID, HCD, and ETD followed by in silico and in vitro analyses for antimicrobial peptide identification. J Proteome Res 14:2649–2658. CrossRefPubMedGoogle Scholar
  3. Baginski ES, Epstein E, Zak B (1972) The measurement of serum total phospholipids. Ann Clin Lab Sci 2:255–267PubMedGoogle Scholar
  4. Barroso RP, Perez KR, Cuccovia IM, Lamy TM (2012) Aqueous dispersions of DMPG in low salt contain leaky vesicles. Chem Phys Lipids 165:169–177. CrossRefPubMedGoogle Scholar
  5. Bittame A, Lopez J, Effantin G, Blanchard N, Cesbron-Delauw M, Gagnon J, Mercier C (2016) Lipid Extraction from HeLa Cells, quantification of Lipids, formation of large unilamellar vesicles (LUVs) by extrusion and in vitro protein-lipid binding assays, Analysis of the incubation product by transmission electron microscopy (TEM) and by flotation across a discontinuous sucrose gradient. Bio Protoc 6:e1963. CrossRefGoogle Scholar
  6. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917. CrossRefPubMedGoogle Scholar
  7. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238–250. CrossRefPubMedGoogle Scholar
  8. Carvajal-Rondanelli P, Aróstica M, Marshall SH et al (2016) Inhibitory effect of short cationic homopeptides against Gram-negative bacteria. Amino Acids 48:1445–1456. CrossRefPubMedGoogle Scholar
  9. Chia CS, Torres J, Cooper M et al (2002) The orientation of the antibiotic peptide maculatin 1.1 in DMPG and DMPC lipid bilayers. Support for a pore-forming mechanism. FEBS Lett 512:47–51. CrossRefPubMedGoogle Scholar
  10. Cutrona KJ, Kaufman BA, Figueroa DM, Elmore DE (2015) Role of arginine and lysine in the antimicrobial mechanism of histone-derived antimicrobial peptides. FEBS Lett 589:3915–3920. CrossRefPubMedPubMedCentralGoogle Scholar
  11. De Leeuw E, Kaat K, Moser C et al (2000) Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase activity. The EMBO J 19:531–541. CrossRefPubMedGoogle Scholar
  12. Epand RM, Vogel HJ (1999) Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta 1462:11–28. CrossRefPubMedGoogle Scholar
  13. Foroutan A, Lazarova T, Padrós E (2011) Study of membrane-induced conformations of substance P: detection of extended polyproline II helix conformation. J Phys Chem B 115:3622–3631. CrossRefPubMedGoogle Scholar
  14. Guzmán F, Marshall S, Ojeda C et al (2013) Inhibitory effect of short cationic homopeptides against gram-positive bacteria. J Pept Sci 19:792–800. CrossRefPubMedGoogle Scholar
  15. Henk WG, Todd WJ, Enright FM, Mitchell PS (1995) The morphological effects of two antimicrobial peptides, hecate-1 and melittin, on Escherichia coli. Scanning Microsc 9:501–507PubMedGoogle Scholar
  16. Hoernke M, Schwieger C, Kerth A, Blume A (2012) Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: complex interplay of electrostatic and hydrophobic interactions. Biochim Biophys Acta Biomembr 1818:1663–1672. CrossRefGoogle Scholar
  17. Hope MJ, Bally MB, Webb G, Cullis PR (1985) Production of large unilamellar vesicles by a rapid extrusion procedure: characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim Biophys Acta 812:55–65. CrossRefPubMedGoogle Scholar
  18. Houghten RA (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc Natl Acad Sci 82:5131–5135. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Jamieson AG, Boutard N, Sabatino D, Lubell WD (2013) Peptide scanning for studying structure-activity relationships in drug discovery. Chem Biol Drug Des 81:148–165. CrossRefPubMedGoogle Scholar
  20. Jorda J, Kajava AV (2010) Protein homorepeats sequences, structures, evolution, and functions. Adv Protein Chem Struct Biol 79:59–88. CrossRefPubMedGoogle Scholar
  21. Lee J, Lee DG (2015) Antimicrobial peptides (AMPs) with dual mechanisms: membrane disruption and apoptosis. J Microbiol Biotechnol 25:759–764. CrossRefPubMedGoogle Scholar
  22. Lee J-K, Park S-C, Hahm K-S, Park Y (2013) Antimicrobial HPA3NT3 peptide analogs: placement of aromatic rings and positive charges are key determinants for cell selectivity and mechanism of action. Biochim Biophys Acta 1828:443–454. CrossRefPubMedGoogle Scholar
  23. Li L, Vorobyov I, Allen TW (2013) The different interactions of lysine and arginine side chains with lipid membranes. J Phys Chem B. 117:11906–21190. CrossRefPubMedGoogle Scholar
  24. Luna OF, Gomez J, Cárdenas C et al (2016) Deprotection reagents in Fmoc solid phase peptide synthesis: moving away from piperidine? Molecules 21(11):1542. CrossRefGoogle Scholar
  25. Mangoni ML, Papo N, Barra D et al (2004) Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli. Biochem J 380:859–865. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Mastroianni JR, Ouellette AJ (2009) Alpha-defensins in enteric innate immunity: functional Paneth cell alpha-defensins in mouse colonic lumen. J Biol Chem 284:27848–27856. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Matsuzaki K, Sugishita K, Harada M et al (1997) Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria. Biochim Biophys Acta 1327:119–130. CrossRefPubMedGoogle Scholar
  28. Mayer LD, Hope MJ, Cullis PR (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858:161–168CrossRefPubMedGoogle Scholar
  29. Mirtič A, Grdadolnik J (2013) The structure of poly-l-lysine in different solvents. Biophys Chem 175:47–53. CrossRefPubMedGoogle Scholar
  30. Monteiro C, Fernandes M, Pinheiro M et al (2015) Antimicrobial properties of membrane-active dodecapeptides derived from MSI-78. Biochim Biophys Acta Biomembr 1848:1139–1146. CrossRefGoogle Scholar
  31. Morrison KL, Weiss GA (2001) Combinatorial alanine-scanning. Curr Opin Chem Biol 5:302–307CrossRefPubMedGoogle Scholar
  32. Mosior M, McLaughlin S (1992) Binding of basic peptides to acidic lipids in membranes: effects of inserting alanine(s) between the basic residues. Biochemistry 31:1767–1773. CrossRefPubMedGoogle Scholar
  33. Nguyen LT, de Boer L, Zaat SAJ, Vogel HJ (2011) Investigating the cationic side chains of the antimicrobial peptide tritrpticin: hydrogen bonding properties govern its membrane-disruptive activities. Biochim Biophys Acta 1808:2297–2303. CrossRefPubMedGoogle Scholar
  34. Phoenix DA, Dennison SR, Harris F (2013) Cationic antimicrobial peptides, in antimicrobial peptides. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. CrossRefGoogle Scholar
  35. Rucker AL, Creamer TP (2002) Polyproline II helical structure in protein unfolded states: lysine peptides revisited. Protein Sci 11:980–985. PubMedPubMedCentralGoogle Scholar
  36. Sato H, Feix JB (2008) Lysine-enriched cecropin-melittin antimicrobial peptides with enhanced selectivity. Antimicrob Agents Chemother 52:4463–4465. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Skerlavaj B, Benincasa M, Risso A et al (1999) SMAP-29: a potent antibacterial and antifungal peptide from sheep leukocytes. FEBS Lett 463:58–62. CrossRefPubMedGoogle Scholar
  38. Soblosky L, Ramamoorthy A, Chen Z (2015) Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy. Chem Phys Lipids 187:20–33. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Sohlenkamp C, Geiger O (2016) Bacterial membrane lipids: diversity in structures and pathways. FEMS Microbiol Rev 40:133–159. CrossRefPubMedGoogle Scholar
  40. Stark M, Liu L-P, Deber CM (2002) Cationic hydrophobic peptides with antimicrobial activity. Antimicrob Agents Chemother 46:3585–3590. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Sviridov DO, Drake SK, Freeman LA et al (2016) Amphipathic polyproline peptides stimulate cholesterol efflux by the ABCA1 transporter. Biochem Biophys Res Commun 471:560–565. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Toke O, Maloy WL, Kim SJ, Blazyk J, Schaefer J (2004) Secondary structure and lipid contact of a peptide antibiotic in phospholipid bilayers by REDOR. Biophys J 87:662–674CrossRefPubMedPubMedCentralGoogle Scholar
  43. Zelezetsky I, Tossi A (2006) Alpha-helical antimicrobial peptides-using a sequence template to guide structure-activity relationship studies. Biochim Biophys Acta Biomembr 1758:1436–1449. CrossRefGoogle Scholar
  44. Zhang X, Oglęcka K, Sandgren S et al (2010) Dual functions of the human antimicrobial peptide LL-37-target membrane perturbation and host cell cargo delivery. Biochim Biophys Acta 1798:2201–2208. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • P. Carvajal-Rondanelli
    • 1
  • M. Aróstica
    • 2
  • C. A. Álvarez
    • 3
    • 4
  • C. Ojeda
    • 5
  • F. Albericio
    • 6
    • 7
  • L. F. Aguilar
    • 8
  • S. H. Marshall
    • 2
    • 5
  • F. Guzmán
    • 2
  1. 1.Escuela de Alimentos, Facultad de Ciencias Agronómicas y de los AlimentosPontificia Universidad Católica de ValparaísoValparaísoChile
  2. 2.Núcleo de Biotecnología de CuraumaPontificia Universidad Católica de ValparaísoValparaísoChile
  3. 3.Laboratorio de Fisiología y Genética Marina (FIGEMA)Centro de Estudios Avanzados en Zonas Áridas (CEAZA)CoquimboChile
  4. 4.Facultad de Ciencias del MarUniversidad Católica del NorteCoquimboChile
  5. 5.Instituto de BiologíaPontificia Universidad Católica de ValparaísoValparaísoChile
  6. 6.Department of Organic Chemistry and CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and NanomedicineUniversity of BarcelonaBarcelonaSpain
  7. 7.School of ChemistryUniversity of KwaZulu-NatalDurbanSouth Africa
  8. 8.Instituto de QuímicaPontificia Universidad Católica de ValparaísoValparaísoChile

Personalised recommendations