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Secondary Structure in de Novo Designed Peptides Induced by Electrostatic Interaction with a Lipid Bilayer Membrane Patrik Nygren,†, Martin Lundqvist,‡,§ Bo Liedberg,† Bengt-Harald Jonsson,*,‡ and Thomas Ederth*,† Division of Molecular Physics and ‡Division of Molecular Biotechnology, IFM, Department of Physics, Chemistry and Biology, Link€ oping University, SE-581 83 Link€ oping, Sweden. §Present address: Center for Molecular Protein Science, Lund University, P.O. Box 124, SE-22100 Lund, Sweden. Present address: Department of Biochemistry & Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104-6059. )
†
Received April 7, 2009. Revised Manuscript Received March 8, 2010 We show that it is possible to induce a defined secondary structure in de novo designed peptides upon electrostatic attachment to negatively charged lipid bilayer vesicles without partitioning of the peptides into the membrane, and that the secondary structure can be varied via small changes in the primary amino acid sequence of the peptides. The peptides have a random-coil conformation in solution, and results from far-UV circular dichroism spectroscopy demonstrate that the structure induced by the interaction with silica nanoparticles is solely R-helical and also strongly pH-dependent. The present study shows that negatively charged vesicles, to which the peptides are electrostatically adsorbed via cationic amino acid residues, induce either R-helices or β-sheets and that the conformation is dependent on both lipid composition and variations in peptide primary structure. The pH-dependence of the vesicle-induced peptide secondary structure is weak, which correlates well with small differences in the vesicles’ electrophoretic mobility, and thus the surface charge, as the pH is varied.
Introduction The interactions of peptides with biological interfaces are studied for a variety of reasons, reflecting the diverse functions of peptides in nature; peptides act as hormones and signaling molecules, they may have antimicrobial properties or act as antifreeze agents, peptides are involved in biomineralization, and the use of peptide antibodies is becoming increasingly popular, to mention but a few. In parallel to these activities, there is growing interest in peptide-surface interactions also from a materials science perspective;1,2 for biomaterials applications such as biocompatibility,3 controlled bioactivity,4 or drug delivery5 but also interactions of peptides with inorganic surfaces, with applications in nanoscale materials fabrication,6,7 photonics,8 molecular electronics,9,10 or cell adhesion.11 The literature on peptides which specifically recognize and bind to various inorganic surfaces is growing rapidly,12,13 and screening techniques *To whom correspondence should be addressed. E-mail:
[email protected] (T.E.);
[email protected] (B.-H.J.). (1) Zanuy, D.; Nussinov, R.; Aleman, C. Phys. Biol. 2006, S80. (2) Ulijn, R. V.; Smith, A. M. Chem. Soc. Rev. 2008, 37, 664. (3) Banwell, E. F.; Abelardo, E. S.; Adams, D. J.; Birchall, M. A.; Corrigan, A.; Donald, A. M.; Kirkland, M.; Serpell, L. C.; Butler, M. F.; Woolfson, D. N. Nat. Mater. 2009, 8, 596–600. (4) Meyers, S. R.; Khoo, X.; Huang, X.; Walsh, E. B.; Grinstaff, M. W.; Kenan, D. J. Biomaterials 2009, 30, 277–286. (5) Branco, M. C.; Schneider, J. P. Acta Biomater. 2009, 5, 817–831. (6) Schiffrin, A.; Riemann, A.; Auw~arter, W.; Pennec, Y.; Weber-Bargioni, A.; Cvetko, D.; Cossaro, A.; Morgante, A.; Barth, J. V. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5279–5284. (7) Sano, K.-I.; Yoshii, S.; Yamashita, I.; Shiba, K. Nano Lett. 2007, 7, 3200– 3202. (8) Zin, M. T.; Munro, A. M.; Gungormus, M.; Wong, N.-Y.; Ma, H.; Tamerler, C.; Ginger, D. S.; Sarikayaa, M.; Jen, A. K.-Y. J. Mater. Chem. 2007, 17, 866. (9) Kitagawa, K.; Morita, T.; Kawasaki, M.; Kimura, S. J. Polym. Sci., Part A: Polym. Chem. 2003, 41, 3493–3500. (10) Sek, S.; Tolak, A.; Misicka, A.; Palys, B.; Bilewicz, R. J. Phys. Chem. B 2005, 109, 18433–18438. (11) Meyers, S. R.; Hamilton, P. T.; Walsh, E. B.; Kenan, D. J.; Grinstaff, M. W. Adv. Mater. 2007, 19, 2492–2498. (12) Brown, S. Nat. Biotechnol. 1997, 15, 269. (13) Evans, J. S.; Samudrala, R.; Walsh, T. R.; Oren, E. E.; Tamerler, C. MRS Bull. 2008, 33, 514.
Langmuir 2010, 26(9), 6437–6448
based on sequence libraries are used in the search for materialsselective sequences (see Evans et al.13 and references therein). Whereas extensive sequence libraries may be created with relative ease, the identification of the effective peptide sequences in these libraries is not straightforward. As far as de novo designed peptides are concerned, much of the design work in this area is based mostly on empirical knowledge, and methods for rational design of selective, surface-binding, or functional peptides are much desired. Although sometimes trends can be discerned in peptide-surface interactions in natural systems (e.g., biomineralinteraction sequences tend to remain unfolded, whereas iceinteraction peptides typically adopt folded structures14), the problem is exacerbated by the complexity of the interactions; the diversity of linear sequences obtainable from the naturally available amino acids is considerable, and since also secondary structure, intrapeptide interactions, and dynamic properties contribute to the interactions with surfaces,15 the interactions between peptides and surfaces may be extremely complicated, and many aspects of the affinity and specificity of peptides binding to surfaces are poorly understood. In previous publications,16,17 we have addressed principles governing the de novo design of peptides forming secondary structure upon interaction with silica nanoparticles. These peptides form helices which are induced and stabilized primarily via attractive and repulsive electrostatic forces. Surface-induced secondary structure has been observed also in subsequent studies of hydroxyapatite18 and functionalized gold nanoparticles.19 The results of these studies aid the basic understanding of protein (14) Evans, J. S. Curr. Opin. Colloid Interface Sci. 2003, 8, 48–54. (15) Skelton, A. A.; Liang, T.; Walsh, T. R. ACS Appl. Mater. Interfaces 2009, 1, 1482–1491. (16) Lundqvist, M.; Nygren, P.; Jonsson, B.-H.; Broo, K. Angew. Chem. 2006, 118, 8349–8353. (17) Nygren, P.; Lundqvist, M.; Broo, K.; Jonsson, B.-H. Nano Lett. 2008, 8, 1844–1852. (18) Capriotti, L. A.; Beebe, T. P.; Schneider, J. P. J. Am. Chem. Soc. 2007, 129, 5281–5287. (19) Fillon, Y.; Verma, A.; Ghosh, P.; Ernenwein, D.; Rotello, V. M.; Chmielewski, J. J. Am. Chem. Soc. 2007, 129, 6676–6677.
Published on Web 03/29/2010
DOI: 10.1021/la100027n
6437
Article
folding, as well as being of interest for the use of peptides in industrial processes and as diagnostic tools, where in both cases the ability to induce and maintain a functional conformation is essential. Using lipid bilayers to study the interaction of peptides and proteins with charged surfaces gives an excellent possibility to understand the cell membrane’s contribution to protein structure and function in living organisms.20-24 It is well-known that lipid membranes, besides providing the necessary environment to native membrane-bound proteins, may induce the formation of amyloid fibrils and that electrostatic interactions contribute significantly to their interactions with the membrane.25-27 Various secondary structure elements have also been found in designed peptides28-30 that interact with vesicles. Further, there is an increasing interest in the understanding of the interaction of lipid bilayers and peptides with antimicrobial activity from a therapeutic point of view, considering the increasing number of antibiotic-resistant bacteria,31-34 and we note that the attachment of many cell-penetrating and antimicrobial peptides to the membrane is mediated via electrostatic interactions. Peptide-bilayer interactions in natural systems show considerable complexity and diversity, and while amphipathic membrane-associating peptides partition into the hydrophobic core of the membrane35 and adopt secondary structure via partitioning-folding coupling,36 there are also examples of electrostatic37 as well as combined electrostatic and hydrophobic attachment of peptides38 and recent examples also of cell-penetrating peptides which rather float on top of membranes than immediately partition into them.39 Thus, it is clear that studies of combined electrostatic peptidemembrane association and secondary structure formation are of potential interest to many areas of biomedical research, and what follows is an expansion of our earlier work on the electrostatic attachment of peptides to silica particles to include the interactions of such peptides with negatively charged lipid bilayers. Herein, we present five de novo designed peptides which were designed to facilitate the formation of an R-helical secondary structure upon adsorption to a negatively charged surface. Three of these peptides form helical structure upon binding to silica (20) Senes, A.; Chadi, D. C.; Law, P. B.; Walters, R. F. S.; Nanda, V.; DeGrado, W. F. J. Mol. Biol. 2007, 366, 436-448; http://degrado.med.upenn.edu/ez. (21) North, B.; Cristian, L.; Fu Stowell, X.; Lear, J. D.; Saven, J. G.; DeGrado, W. F. J. Mol. Biol. 2006, 359, 930–939. (22) Lind, J.; R€am€o, T.; Rosen Klement, M. L.; Barany-Wallje, E.; Epand, R. M.; Epand, R. F.; M€aler, L.; Wieslander, A˚. Biochemistry 2007, 46, 5664–5677. (23) van Meer, G.; Voelker, D. R.; Feigenson, G. W. Nat. Rev. Mol. Cell Biol. 2008, 9, 112–124. (24) Xie, K.; Dalbey, R. E. Nat. Rev. Microbiol. 2008, 6, 234–244. (25) Terzi, E.; Holzemann, G.; Seelig, J. Biochemistry 1997, 36, 14845–14852. (26) Olofsson, A.; Borowik, T.; Gr€obner, G.; Sauer-Eriksson, A. E. J. Mol. Biol. 2007, 374, 186–194. (27) Miura, T.; Yoda, M.; Takaku, N.; Hirose, T.; Takeuchi, H. Biochemistry 2007, 46, 11589–11597. (28) Meier, M.; Seelig, J. J. Am. Chem. Soc. 2007, 130, 1017–1024. (29) Vagt, T.; Zsch€ornig, O.; Huster, D.; Koksch, B. ChemPhysChem 2006, 7, 1361–1371. (30) Kiyota, T.; Lee, S.; Sugihara, G. Biochemistry 1996, 35, 13196–13204. (31) Huang, H. W. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 1292–1302. (32) Wieprecht, T.; Apostolov, O.; Beyermann, M.; Seelig, J. Biochemistry 2000, 39, 442–452. (33) Zasloff, M. Nature 2002, 415, 389–395. (34) Strøm, M. B.; Rekdal, Ø.; Svendsen, J. S. J. Pept. Sci. 2002, 8, 431–437. (35) White, S. H.; Wimley, W. C. Annu. Rev. Biophys. Biomol. Struct. 1999, 28, 319–365. (36) Wimley, W. C.; White, S. H. Nat. Struct. Biol. 1996, 3, 842–848. (37) Ben-Tal, N.; Honig, B.; Peitzsch, R. M.; Denisov, G.; McLaughlin, S. Biophys. J. 1996, 71, 561–575. (38) Bonev, B.; Watts, A.; Bokvist, M.; Gr€obner, G. Phys. Chem. Chem. Phys. 2001, 3, 2904. (39) Ciobanasu, C.; Harms, E.; T€unnemann, G.; Cardoso, M. C.; Kubitscheck, U. Biochemistry 2009, 48, 4728–4737.
6438 DOI: 10.1021/la100027n
Nygren et al.
surfaces, which are characterized essentially by having negatively charged groups in static positions on their surfaces. In the present study, however, we focus on the interaction with vesicles, in which the content of the negatively charged lipid 1,2-dioleoyl-snglycero-3-phosphoglycerol (PG) is varied from 0 to 40%. Notably, the surface of the vesicles is dynamic because the lipids in the vesicles are free to diffuse laterally in the bilayer, implying that the charged residues can rearrange on the vesicle surface. By using circular dichroism (CD) spectroscopy, it was possible to monitor the effects of moderate changes in peptide primary sequence on the peptide secondary structure, as the peptides were introduced to lipid bilayer vesicles of various compositions. Interestingly, two of the peptides form β-structures on vesicles with high PG content but form no defined secondary structure upon interaction with silica nanoparticles. Furthermore, the effect of peptide adsorption on apparent vesicle size was investigated with dynamic light scattering (DLS), the variation in vesicle surface charge with varying anionic lipid content was monitored by electrokinetic measurements, and the energetics of the peptide-membrane interactions were monitored via isothermal titration calorimetry (ITC) for some of the peptides.
Materials and Methods Chemicals. Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-LAsp(OtBu)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, and Fmoc-L-Tyr(tBu)-OH were purchased from Novabiochem (Darmstadt, Germany). Fmoc-PAL-PEG-PS was purchased from Applied Biosystems (Foster City, CA). Trifluoroacetic acid (TFA) and chloroform were purchased from Merck (Darmstadt, Germany). Triisopropylsilane (TIS), diethylether, and ethanedithiol (EDT) were purchased from SigmaAldrich (Schnelldorf, Germany). 1,2-Dioleoyl-sn-glycero-3-phosphocholine (PC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (PG) were obtained from Larodan (Malm€ o, Sweden), cholesterol (Chol) was from Sigma-Aldrich (Schnelldorf, Germany), and all were used as received. Peptide Synthesis. Syntheses of the five peptides were performed on a Pioneer peptide synthesis system. Fmoc-PAL-PEGPS was used as solid support. Each coupling cycle was set to 2 h. All peptides except R2X were cleaved from the resin using TFA/ TIS/H2O (95/2.5/2.5), and peptide R2X was cleaved using TFA/ TIS/H2O/EDT (94/2.5/2.5/1). The peptides were precipitated with diethylether and centrifuged. The pellets were dissolved in Milli-Q water, frozen with liquid nitrogen, and lyophilized. Peptides were purified with a C8 reversed phase HPLC (Dynamax solvent delivery system model SD-1) with a diode-array UVdetector (Dynamax absorbance detector model UV-1). The purity of the peptides was confirmed by MALDI-ToF-MS (PerSeptive Biosystems Voyager - DE STR BioSpectrometry Workstation). Vesicle Preparation. Neutral vesicles were prepared using pure PC or PC/cholesterol (Chol) mixtures in a 60/40 ratio (all ratios refer to mol fractions), and negatively charged vesicles were prepared using increasing amounts of PG as the anionic component in Chol/PC/PG mixtures prepared to the following fractions: 40/58/2, 40/50/10, 40/40/20, and 40/20/40 (see Figure 1). Lipids were dissolved in chloroform and then mixed to the desired molar ratios in glass vials. A thin lipid film was formed on the walls of the vials by removing the chloroform in a stream of nitrogen gas while rotating the vial. The lipids were then dried for at least 1 h in a vacuum system at
