pubs.acs.org/Langmuir © 2010 American Chemical Society
Peptide Adsorption to Cyanine Dye Aggregates Revealed by Cryo-Transmission Electron Microscopy Hans v. Berlepsch,*,† Enrico Brandenburg,‡ Beate Koksch,‡ and Christoph B€ottcher† †
Forschungszentrum f€ ur Elektronenmikroskopie, Institut f€ ur Chemie und Biochemie, Freie Universit€ at Berlin, Fabeckstrasse 36 a, D-14195 Berlin, Germany, and ‡Institut f€ ur Chemie und Biochemie, Freie Universit€ at Berlin, Takustrasse 3, D-14195 Berlin, Germany Received March 9, 2010. Revised Manuscript Received March 30, 2010 The binding interaction between aggregates of the 5-chloro-2-[[5-chloro-3-(3-sulfopropyl)-3H-benzothiazol-2-ylidene]methyl]-3-(3-sulfopropyl)benzothiazolium hydroxide inner salt ammonium salt (CD-1) and R-helix, as well as β-sheet forming de novo designed peptides, was investigated by absorption spectroscopy, circular dichroism spectroscopy, and cryogenic transmission electron microscopy. Both pure dye and pure peptides self-assembled into well-defined supramolecular assemblies in acetate buffer at pH=4. The dye formed sheetlike and tubular H- and J-aggregates and the peptides R-helical coiled-coil assemblies or β-sheet rich fibrils. After mixing dye and peptide solutions, tubular aggregates with an unusual ultrastructure were found, most likely due to the decoration of dye tubes with monolayers of peptide assemblies based on the strong electrostatic attraction between the oppositely charged species. There was neither indication of a transfer of chirality from the peptides to the dye aggregates nor the opposite effect of a structural transfer from dye aggregates onto the peptides secondary structure.
Introduction Supramolecular assemblies of dye molecules1 have attracted much attention in recent years due to their unique photophysical properties, which make them interesting for technical application in the field of optoelectronics,2,3 as models for light-harvesting photosynthetic antenna complexes,4,5 as structure-sensitive probes in bioanalytics,6,7 and for the development of functional nanomaterials.8 Cyanine dyes are particularly interesting organic dyes because they can form supramolecular aggregates. Because of the exceptionally strong interaction between the monomers’ transition dipole moments and the high degree of positional order,9 cyanine dyes can form so-called J-aggregates, which are characterized by a dramatic red shift and narrowing of their lowest electronic energy transition relative to the dye monomer.10 J-aggregates of cyanine dyes are formed in polar solvents upon increasing the concentration above a critical level. Besides these neat J-aggregates, a variety of complex nanostructures have been reported to form with the aid of templates onto which dye *To whom correspondence should be addressed: e-mail berlepsc@chemie. fu-berlin.de. (1) W€urthner, F. Supramolecular Dye Chemistry; Top. Curr. Chem. Vol. 258; Springer: Berlin, 2005. (2) Kobayashi, T. J-Aggregates; World Scientific: Singapore, 1996. (3) Hoeben, F. J. M.; Jonkheijm, P.; Meijer, E. W.; Schenning, A. P. H. J. Chem. Rev. 2005, 105, 1491–1546. (4) Sautter, A.; Kaletas, B. K.; Schmid, D. G.; Dobrawa, R.; Zimine, M.; Jung, G.; van Stokkum, I. H. M.; De Cola, L.; Williams, R. M.; W€urthner, F. J. Am. Chem. Soc. 2005, 127, 6719–6729. (5) Kirstein, S.; Daehne, S. Int. J. Photoenergy 2006, 5, 1-21. (6) Volkova, K. D.; Kovalska, V. B.; Balanda, A. O.; Vermeij, R. J.; Subramaniam, V.; Slominskii, Yu. L.; Yarmoluk, S. M. J. Biochem. Biophys. Methods 2007, 70, 727–733. (7) Kitts, C. C.; Vanden Bout, D. A. J. Phys. Chem. B 2009, 113, 12090–12095. (8) Varghese, R.; Wagenknecht, H.-A. Chem. Commun. 2009, 2615–2624. (9) Czikkely, V.; F€orsterling, H. D.; Kuhn, H. Chem. Phys. Lett. 1970, 6, 207– 210. (10) (a) Scheibe, G. Angew. Chem. 1937, 50, 51–51. (b) Jelly, E. E. Nature 1936, 138, 1009–1010. (11) Peyratout, C.; Daehne, L. Phys. Chem. Chem. Phys. 2002, 4, 3032. (12) Miyagawa, T.; Yamamoto, M.; Muraki, R.; Onouchi, H.; Yashima, E. J. Am. Chem. Soc. 2007, 129, 3676–3682.
11452 DOI: 10.1021/la100944d
molecules spontaneously aggregate. Various synthetic polymers11,12 and biopolymers, such as polypeptides,13-15 polysaccharides,16 and DNA,17 have been used as templates. While the spectroscopic properties of all the different dye assemblies have been well documented, their detailed supramolecular structures in solution have not been sufficiently characterized. This is due to the smallness of many of these aggregates and a lack of suitable experimental techniques. For some neat J-aggregates, however, the supramolecular organization on the nanometer-to-micrometer scale have recently become available.5,18-24 This was achieved by using cryogenic transmission electron microscopy (cryo-TEM), a technique that allows direct high-resolution imaging of organized assemblies in the native environment of the solvent. For the present study the cryo-TEM technique was used to characterize the more complex superstructures of a cyanine dye formed in the presence of a template. As template we utilized two 26-residue de novo designed peptides. The design is based on the naturally occurring coiled-coil (13) Stryer, L.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 1411–1418. (14) Pasternack, R. F.; Giannetto, A.; Pagano, P.; Gibbs, E. J. Am. Chem. Soc. 1991, 113, 7799–7800. (15) Zhang, Y.; Xiang, J.; Tang, Y.; Xu, G.; Yan, W. Chem. Phys. Chem. 2007, 8, 224–226. (16) Kim, O.-K.; Je, J.; Jernigan, G.; Buckley, L.; Whitten, D. J. Am. Chem. Soc. 2006, 128, 510–516. (17) Wang, M.; Silva, G. L.; Armitage, B. A. J. Am. Chem. Soc. 2000, 122, 9977– 9986. (18) von Berlepsch, H.; B€ottcher, C.; D€ahne, L. J. Phys. Chem. B 2000, 104, 8792–8799. (19) von Berlepsch, H.; B€ottcher, C.; Ouart, A.; Burger, C.; D€ahne, S.; Kirstein, S. J. Phys. Chem. B 2000, 104, 5255–5262. (20) von Berlepsch, H.; Regenbrecht, M.; D€ahne, S.; Kirstein, S.; B€ottcher, C. Langmuir 2002, 18, 2901–2907. (21) von Berlepsch, H.; B€ottcher, C.; Ouart, A.; Regenbrecht, M.; Akari, S.; Keiderling, U.; Schnablegger, H.; D€ahne, S.; Kirstein, S. Langmuir 2000, 16, 5908– 5916. (22) von Berlepsch, H.; Kirstein, S.; B€ottcher, C. J. Phys. Chem. B 2004, 108, 18725–18733. (23) von Berlepsch, H.; Kirstein, S.; Hania, R.; Pugzlys, A.; B€ottcher, C. J. Phys. Chem. B 2007, 111, 1701–1711. (24) Vlaming, S. M.; Augulis, R.; Stuart, M. C. A.; Knoester, J.; van Loosdrecht, P. H. M. J. Phys. Chem. B 2009, 113, 2273–2283.
Published on Web 04/05/2010
Langmuir 2010, 26(13), 11452–11460
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Article
Scheme 1. (a) Structural Formula of Thiacarbocyanine CD-1 and (b) Sequences of Model Peptides 1 and 2
Figure 1. Absorption spectra of aqueous CD-1 solutions at dif-
folding motif in which the primary peptide sequence is characterized by a periodicity of seven residues (cf. Figure S1 of the Supporting Information). In one case the sequence was chosen to give a stable R-helical conformation independent of the pH of the solvent. The second peptide contains additional solventexposed valine residues, which enhance the propensity to form β-sheets. Depending on pH, three different conformations, namely random coil, R-helical, and β-sheet, were observed for the same peptide sequence. A stable β-sheet conformation was adopted after 1 day at acidic pH conditions. The morphology of the peptide aggregates has been well investigated in separate studies,25,26 which should allow us to make direct comparisons with the more complex dye-peptide assemblies. As dye, we chose the anionic thiacarbocyanine CD-1 (cf. Scheme 1), which was expected to strongly interact with the peptides due to their positive net charge under acidic pH conditions. An additional intriguing feature that is often observed in polymer-templated aggregation of dyes is the transfer of chirality from the template to the dye aggregate.13-16,27,28 Recently, we showed that the helicity of tubular J-aggregates composed of achiral amphiphilic dye molecules could be controlled by chiral alcohols.29 For the present achiral dye CD-1 we expected similar effects to be induced by the chiral peptides. In this paper, we discuss the spectroscopic properties of the CD-1 dye aggregates in the presence of β-sheet forming and R-helical peptides 1 and 2, respectively, for different dye/peptide ratios and at different preparation conditions. The main goal was the thorough characterization of the obtained aggregate structures by cryo-TEM. Cryo-TEM reveals highly structured sheetlike and tubular assemblies several hundreds of nanometers in size with an unusual modification of the tube surface. Comparison with the morphologies of aggregates of pure dye suggests that these assemblies represent peptide-covered dye aggregates.
Experiment Materials and Sample Preparation. The 5-chloro-2-[[5chloro-3-(3-sulfopropyl)-3H-benzothiazol-2-ylidene]methyl]3-(3-sulfopropyl)benzothiazolium hydroxide inner salt ammonium salt (CD-1) was supplied by FEW Chemicals (Wolfen, Villa, A.; Vagt, T.; Koksch, B.; Mark, A. E. Biophys. J. 2005, (25) Pineiro, A.; 89, 3701–3713. (26) (a) Pagel, K.; Wagner, S. C.; Samedov, K.; von Berlepsch, H.; B€ottcher, C.; Koksch, B. J. Am. Chem. Soc. 2006, 128, 2196–2197. (b) Pagel, K.; Wagner, S. C.; Rezaei Araghi, R.; von Berlepsch, H.; B€ottcher, C.; Koksch, B. Chem.;Eur. J. 2008, 14, 11442–11451. (27) Slavnova, T. D.; G€orner, H.; Chibisov, A. K. J. Phys. Chem. B 2007, 111, 10023–10031. (28) Yang, Q.; Xiang, J.; Li, Q.; Yan, W.; Zhou, Q.; Tang, Y.; Xu, G. J. Phys. Chem. B 2008, 112, 8783–8787. (29) von Berlepsch, H.; Kirstein, S.; B€ottcher, C. J. Phys. Chem. B 2003, 107, 9646–9654.
Langmuir 2010, 26(13), 11452–11460
ferent dye concentrations: (black) 6.77 10-4 M, (blue) 1.39 10-3 M, (red) 2.74 10-3 M. Normalized absorption spectrum of CD-1 monomers in MeOH (green). Monomer (M), H-aggregate (H), and J-aggregate (J) bands are labeled.
Germany) and was used as received. The molecular mass is 612.6 g/mol. The molar extinction coefficient in methanol is ε=8.2 104 L/(mol cm). 1.0 10-4 M dye solutions were prepared by direct dissolving the dye in acetate buffer (10 mM, pH= 4) or by dilution of stock solutions (1.0 10-3 M dye dissolved in Millipore water or MeOH) with acetate buffer. Peptides were synthesized on a Multi-Syntech Syro XP peptide synthesizer at solid phase using the Fmoc strategy and FmocLeu-OWang resin (0.65 mmol/g). For UV concentration determination, the peptides were N-terminally labeled with anthranilic acid (Abz). The peptides were cleaved from the resin by reaction with 4 mL solution containing 10% (w/v) triisopropylsilane, 1% (w/v) water, and 89% (w/v) trifluoroacetic acid (TFA). The crude peptides were purified by reversed-phase HPLC on a Knauer smartline manager 5000 system equipped with a C8 (10 μm) LUNA Phenomenex column. The peptides were eluted with a linear gradient of acetonitrile/water/0.1% TFA and identified by MALDI-TOF MS. All MS analyses were performed using a Bruker Reflex III spectrometer with TOF mass analyzer. Peptide purity was determined by analytical HPLC on a Merck LaChrom system equipped with a C8 (10 μm) LUNA Phenomenex column. The used gradient was similar to those of the preparative HPLC. The peptide solutions were prepared in freshly filtered acetate buffer (10 mM, pH=4). Fibrils were formed at room temperature under stationary conditions. The peptide concentration was calculated by comparing the absorbance at 325 nm with a calibration curve determined by the absorbance of Abz-glycine at different concentrations in the corresponding buffer. Structure formation was checked by CD spectroscopy. Methods. The isotropic absorption spectra were measured with a Lambda 9 spectrophotometer (Perkin-Elmer) and the circular dichroism (CD) spectra with a J-810 spectropolarimeter (Jasco Corp.) at 21 C. TEM was performed using a Philips CM12 transmission electron microscope at 100 kV and a primary magnification of 58,300. Cryo-TEM sample grids were plunged into liquid ethane at its melting point for vitrification and imaged at T=-178 C. The defocus for cryo-TEM was chosen to be 1.5 μm. For staining electron microscopy, aliquots of solutions were first absorbed on carbon-coated collodium films placed on copper grids. After blotting and staining with 1% phosphotungstic acid (PTA) the grids were air-dried. Further details can be found in ref 22.
Results and Discussion Neat CD-1 Solutions. The absorption spectrum of the dye CD-1 in MeOH (Figure 1) exhibited a monomer peak (M) centered at a wavelength of 429 nm and a small vibrionic shoulder at 406 nm.11 In aqueous solution the spectra depend strongly on the concentration. The monomer peak became weaker with increasing DOI: 10.1021/la100944d
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Figure 2. Absorption (above, left ordinate) and CD (below, right ordinate) spectra of 4.0 10-5 M dye solutions in acetate buffer (10 mM, pH = 4) 1 day after preparation from stock solutions (broken lines) of 1.0 10-3 M in MeOH (black), 1.0 10-3 M in water (red), or 1.0 10-4 M in acetate buffer (10 mM, pH = 4) (blue).
concentration while new peaks centered at 406 and 464 nm evolve, which are attributed to the formation of aggregates. The aggregates with a hypsochromic absorption shift relative to the monomer band (at 406 nm) are termed H-aggregates, while those with a bathochromic shift (at 464 nm) are termed J-aggregates. H-aggregation occurs when the adjacent monomers forming an assembly are only slightly offset from one another and J-aggregation when the offset is large.30 Aggregation of CD-1 started at a very low dye concentration of
