J. Phys. Chem. B 2008, 112, 8783–8787
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Chiral Transformation of Cyanine Dye Aggregates Induced by Small Peptides Qianfan Yang,†,‡ Junfeng Xiang,† Qian Li,†,‡ Wenpeng Yan,§ Qiuju Zhou,†,‡ Yalin Tang,*,† and Guangzhi Xu† Beijing National Laboratory for Molecular Sciences (BNLMS), Center for Molecular Sciences, State Key Laboratory for Structural Chemistry for Unstable and Stable Species, Institute of Chemistry, Chinese Academy of Sciences (ICCAS), Graduate UniVersity of Chinese Academy of Sciences, and Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China ReceiVed: April 9, 2008
Three small peptides (K4, K5, and K6) with different length were designed to induce the transformation of the assembled state and the chirality of cyanine dye supramolecule. The absorption and circular dichroism (CD) results indicated that, the peptides tend to induce cyanine dye to H-aggregation, competed with Na+ in PBS, which would induce dye to J-aggregation. Meanwhile, all three peptides could influence the chirality of both J-aggregates induced by Na+ and H-aggregates, among which K6 could induce chiral reversion of J-aggregates. Furthermore, molecular modeling and energy calculation results have shown that the peptides with different chain length have different conformations. This might be the reason for cyanine dye to form the different chiral assembly induced by these oligo-peptide templates. 1. Introduction Chiral supramolecules have been a topic of keen interest in scientific community for decades based on their novel properties and wide applications.1 Compared with achiral supramolecules, chiral supramolecules exhibit many special behaviors which enable them to be a unique class of novel materials.2 Being a large class of fluorescent pigment, cyanine dye has been used extensively as photosensitizers in color photography for centuries.3 With the discovery of the novel photophysical and photochemical properties, they have been applied in numerous fields, such as nonlinear optics,4 biological fluorescent detection,5 biomedicine,6 and photodynamic therapy of tumor.7 In solution, cyanine dyes can spontaneously assemble to aggregates by varying the solvent or adding salt.8 The aggregates can be assembled in various forms, for example, H- or J-aggregates.9 Such assembly properties have been explained qualitatively by using the exciton model.10 Additionally, the aggregates are induced to present chirality when dye molecules self-assemble on unsymmetrical or chiral template.11 Recently, many researchers focus on assembling chiral supramolecule from achiral cyanine dye templated by many biomacromolecules, such as DNAs,11b,12 proteins,13 polypeptides,14 lipid assemblies,15 and amyloses.1b Besides many efforts on assembling chiral supramolecules with either negative or positive chirality,1b,11b,16 chirality transformation of cyanine dye Jaggregates has also been achieved by using protein as template.17 However, templates reported previously are mainly concerned with macromolecules, while chiral supramolecular assembly induced by small molecule is less documented. In this paper, three peptides with different chain length (K4, K5, and K6) were * To whom correspondence should be addressed. Tel: +86-10-62522090. Fax: +86-10-6252-2090. Email:
[email protected]. † Beijing National Laboratory for Molecular Sciences (BNLMS), Center for Molecular Sciences, State Key Laboratory for Structural Chemistry for Unstable and Stable Species, Institute of Chemistry, Chinese Academy of Sciences (ICCAS). ‡ Graduate University of Chinese Academy of Sciences. § Technical Institute of Physics and Chemistry, Chinese Academy of Sciences.
CHART 1: Molecular Formulas of Cyanine Dye MTC and the Peptides K4, K5, and K6
designed to interact with cyanine dye MTC [3,3′-di(3-sulfopropyl)-4,5,4′,5′-dibenzo-9-methyl-thiacarbocyanine triethylammonium salt] (the molecular formulas are shown in Chart 1). These peptides have the following features: (1) having only one kind of side-chain functional group; (2) possessing only one kind of charge (according to the simulative pI titration curve obtained from DNAStar software, shown in Supporting Information, Figure S1. Under the experimental pH value, each side chain of the peptides contains one cation); and (3) having simple conformation due to short chain. Interestingly, the three peptides could affect the transformation of dye assembled states together with their chirality, as natural biomacromolecule does.1a,11b,17 The interaction mechanism was discussed, indicating that the conformation of the small peptides could strongly influence MTC assembly. Different orientation of the side chains of the peptides owing to different chain length plays very important roles in dye aggregation. Peptide with wellseparated side chains would facilitate the H-aggregation, and
10.1021/jp803076d CCC: $40.75 2008 American Chemical Society Published on Web 06/27/2008
8784 J. Phys. Chem. B, Vol. 112, No. 29, 2008
Figure 1. The absorption spectra of 5 µM MTC with different concentrations of the peptides in PBS: (a) K4, (b) K5, (c) K6. The dash line is the absorption spectrum of 4 µM MTC in methanol solution, which is in monomer state.
in certain condition, it could also induce the chirality transformation of dye aggregations. 2. Experimental Details 2.1. Materials and Sample Preparation. The cyanine dye (MTC) was synthesized according to Hamer18 and Fichen’s19 methods, and the purity was proved by MS, elemental analysis, and NMR (data are shown in Supporting Information, Section Sb). The peptides K4, K5, and K6 (purity 98%) were purchased from Xi’an HuaChen Biotech Co. Ltd. All of the peptides were used without further purification. Analytical grade methanol was purchased from Beijing Chem. Co. The stock solution of MTC was prepared by dissolving the compound directly in methanol. The stock solutions of the peptides were prepared by dissolving a certain amount of sample directly into phosphate buffer solution (PBS, 10 mM NaH2PO4/ Na2HPO4, pH 5.5). The measured sample was prepared by adding a quantity of the peptide solution into MTC solution
Yang et al.
Figure 2. The CD spectra of 5µM MTC H-band with different concentrations of the peptides in PBS. (a) K4, (b) K5, (c) K6.
and then diluted to certain volume by PBS. The sample solutions were kept in darkness overnight at room temperature before measurement in order to realize the complete interaction of MTC with peptide. 2.2. Absorption and CD Spectra Experiments. The absorption and CD spectra were obtained in 10 mm quartz cells from a UV-1601PC spectrophotometer and a JASCO J-815 spectropolarimeter, respectively. Double-distilled water was used for all absorption and CD measurements. All experiments were performed in PBS (10 mM NaH2PO4/Na2HPO4, pH 5.5). 2.3. Molecular Modeling and Energy Calculation of the Small Peptides. The conformations of K4, K5, and K6 were built on a SGI workstation by using builder module of Insight II 2005 software and the charge was calculated by using the Amber’s Antechamber module.20 Then, the UCSF Chimera software with Amber force field was carried out to optimize the structures using the steepest descent method for 10 000 steps with a step size of 0.02 Å. Finally, the potential energy of the three peptides was evaluated.
Chiral Transformation Induced by Small Peptides
Figure 3. The CD spectra of 5 µM MTC J-band with different concentrations of the peptides in PBS. (a) K4, (b) K5, (c) K6. The dash line is the CD signal of MTC J-aggregates assembled in PBS without the peptides.
3. Results and Discussion 3.1. Absorption Spectra. In methanol solution, MTC mainly exists in monomer state (as shown by dash line in Figure 1a. When PBS is added into MTC solution, MTC exhibits only a predominant absorption band assigned to J-aggregate. This tendency to J-aggregation could be due to the salt effect and the hydrophobic nature of MTC molecules.9 However, as shown in Figure 1, adding the peptides into MTC solution and then diluting by PBS led to substantial spectral changes: a gradual decrease in the absorbance of J-aggregate accompanied with the appearance of two new peaks located around 489 nm (489, 490.5, and 487 nm for K4, K5, and K6, respectively) and 444 nm, which could be assigned to H-aggregates.9 Further increasing the concentration of the peptides caused the absorbance of J-aggregates gradually decreasing while that of H-aggregates increasing simultaneously. Obviously, there exists a competition between H- and J-aggregation from MTC monomer. Compared with J-ag-
J. Phys. Chem. B, Vol. 112, No. 29, 2008 8785 gregates, the molecular arrangement in H-aggregates is more regular and the distance among dye molecules is shorter.9 In the case of the small peptides, although they play the role of cation centers (neutralizing electrostatic repulsion between dye molecules, attracting and gathering them) as Na+ does, the distance among the cations is shorter than that of Na+, which distribute into solution more random, since the small peptides carry more than one cation in one molecule. Consequently, the small peptides could attract dye molecules closer to each other and induce assembly of H-aggregates, rather than J-aggregates. Furthermore, different phenomena were observed in aggregation by adding different peptides under the similar condition. K4 or K6 can induce MTC H-aggregation completely without any J-aggregates assembled (K4 at the concentration ratio [K4]/ [MTC] ) 0.28/5, while K6 at the concentration ratio [K6]/ [MTC] ) 0.8/5), while K5 cannot. It is inferred that K4 has the strongest ability to induce MTC assembling to H-aggregates, K6 takes the second place, and K5 is third. The results are a little surprising because all peptides contain only one kind of amino acid residue lysine, which carry one cation on each sidechain. Electrostatics is the primary force of the interaction between MTC molecule and the peptides. If the electrostatic interaction existed merely, K6 would carry the most cations and should have the strongest ability to induce MTC H-aggregation. However, it is not the fact. We realized that there is something different existing in the interaction between the small peptides and MTC besides electrostatic interaction, maybe the structural factor. 3.2. Induced Chirality of MTC H- and J-Aggregates. When a supramolecule is formed under unsymmetrical or chiral conditions, the supramolecule will exhibit certain chirality. So in order to make clear the functions of the molecular conformations of the templated peptides, circular dichroism (CD) measurements of both MTC H- and J-aggregates were carried out. It is found that both MTC H- and J-aggregation processes are perturbed by the peptides, dye arrangements in them are induced to certain orientation, and finally both the induced Hand J-aggregates present distinct chiralities. H-Aggregates. Since H-aggregates of MTC are totally formed by the addition of peptides, the dye molecular arrangement in them must be influenced by the backbone orientation of the peptides. Figure 2 shows the CD spectra of H-aggregates without and with the peptides. As we see, all H-aggregates presented CD signals and different CD signals of MTC H-band were observed with different peptides. For K4, adding K4 into MTC solution led to a trisignate CD signal: a negative peak in the middle at 487.5 nm and two positive peaks at 464.5 and 523 nm, respectively. Further addition of K4 resulted in complicated changes: the two positive signals were both transformed to negative, together with blue shift about 4-6 nm; while the intensity of the negative signal decreased coupled with blue shift about 8 nm. In the case of K5, the presence of K5 induced a quadrusignate CD signals: a positive peak in the middle at 481 nm and three negative peaks at 437, 462, and 520.5 nm, respectively. Further increasing the concentration of K5 resulted in the enhancement of all signals’ intensities, coupled with a slight red shift. As for K6, adding K6 into MTC solution led to a trisignate CD signal: a negative peak in the middle at 481.5 nm and two positive peaks at 459 and 520.5 nm, respectively. Further addition of K6 resulted in greatly increasing of all the relative intensity of the CD signals. We noticed that the longer the peptide chain is, the more regular the induced CD signals represent. The H-aggregates CD signals in the present of K5 are not so regular as that in the
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Figure 4. The optimal conformations of the peptides. The backbones display in ball-and-stick style while the side chains in line style. All the protons were removed.
present of K6. Moreover, the H-aggregates CD signals induced by K5 are totally opposite to that induced by K6 and their intensity is much weaker (only about one tenth of that with K6). H-band in the present of K4 is more irregular, indicating different kinds of H-aggregates are involved. This may be also caused by the distinct conformations of the peptides. J-aggregates. Besides H-aggregates, MTC J-aggregates also present unique chiral transformation with peptides. As shown in Figure 3, when MTC solution was diluted by PBS, a bisignate CD signal, a negative first Cotton Effect at longer wavelength and a positive second one at shorter wavelength centered at 661.5 nm, corresponding to left-hand helix,21 is observed. This indicates MTC could assemble to chiral J-aggregates in the effect of Na+. Adding different peptides into the MTC solution resulted in different CD spectra of MTC J-band. For K4, the bisignate CD signal of MTC J-band was transformed into a positive CD band at 662.5 nm when K4 is present. The lack of splitting in the J-band indicates that dye molecules were in the form of isolated oligomers (dimers or trimers) rather than continuous aggregates, and that there was not electronic dislocation between adjacent oligomers.11b Adding of K5 to MTC solution resulted in the decrease of the CD signals’ intensities with a 4 nm blue shift in the center. Interestingly, addition of K6 into MTC solution led to a total chiral transformation which exhibited bisignate CD signal (with a positive first Cotton Effect at longer wavelength and a negative second one at shorter wavelength centered at 652.5 nm) coupled with blue shift about 9 nm, compared with the previous left-handed helix formed in MTC PBS solution. Clearly, the observed changes indicate that MTC J-aggregates have different packing modes with and without K6. For all peptides, further increasing their concentration in MTC solution only decreased the CD signals’ intensities, due to the stronger tendency to H-aggregation in the presence of the peptides, as seen in the absorption spectra. These results are a little surprising. As is previously reported,1a the induced CD spectra of porphyrin J-aggregates exhibit different chirality when they bind to poly-L-glutamate with R-helical and random-coil conformation, respectively.1b Our previous research17,22 on the interaction between human serum albumin (HSA) and cyanine dye PTC (whose structure is very similar as MTC) has shown that the induced J-aggregates could exhibit left-handed or right-handed chirality depending on the relative concentration ratio of [HSA]/[dye], accompanied with disassembly of them to monomers. Such chiral reversion could be assigned to the binding of J-aggregates to R-helix or random coil part in HSA. However, in the case of the peptides with short chain here, they do not exhibit any well-ordered secondary structure as natural proteins do, as shown in Figure 4. Thus,
they do not have pocketlike interspace to accommodate a single dye molecule as HSA does. Furthermore, since each amino acid residue in peptide has a positive charge, every residue may attract one close-by monomer by electrostatic interaction, which facilitates the close packing of dye molecules and consequent assembling of H-aggregates. This may be the reason why monomer is observed in the presence of HSA while only H-aggregates appeared in the presence of peptides with short chain. However, a question still exists that why H- and J-aggregates exhibit different chiralities in the presence of peptides with slightly different chain length. Possibly, the chain length of the peptides may influence the peptides’ conformation which affects the interaction of the dye molecules among aggregates. In order to understand this issue, the optimal conformations of the peptides have been created by using molecular modeling method (as shown in Figure 4). Clearly, the optimal conformations of these peptides are totally different. K4 has a relatively more extended conformation while all side chains separate apart from each other due to electrostatic interaction. The surface cations distribute symmetrically and steric hindrance is relatively small. Therefore, it would be easier for MTC molecule to be attracted and inserted into the vacancies between side chains without strong resistance from the nearby residues, which may facilitate the H-aggregation. This result is consistent with the fact that MTC induced nearly full assembly to H-aggregates with the lowest concentration ratio of [K4]/[MTC] (about 0.28/ 5), as well as chiral transformation (about range from 0.04/5 to 0.12/5). Because the side chains of K4 are well-separated, the MTC molecules in aggregates should be a little far away from each other and electronic dislocation could not transfer between dye aggregates, which could explain why the induced Jaggregates present an unsplit CD signal at higher [K4]. For K5, the conformation of K5 is slightly tense, and the two residues at each end are relatively closer to each other, making it difficult for MTC molecule to insert. Consequently, the complete assembly to H-aggregates or chiral transformation could not be observed because of strong steric hindrance. The conformation of K6 is also extended but not so much as that of K4. Therefore, only at a relatively higher concentration ratio of [K6]/[MTC] it can induce the full assembly to H-aggregates (about 0.8/5) and chirality transformation (about range from 0.1/5 to 0.3/5). The single point energies of the peptides (as shown in Table 1) calculated by UCSF Chimera software also provide similar results. The energy of K5 is the highest among the peptides while K4 and K6 whose side chains are apart from each other have relatively lower energies.
Chiral Transformation Induced by Small Peptides TABLE 1: The Single Point Energies of the Peptides Calculated by UCSF Chimera Software peptide
calculated single point energy (Kcal · mol-1)
K4 K5 K6
178.261 212.661 152.585
3. Conclusion The structure and chirality of cyanine dye in the presence of small peptides with different length have been investigated. It was found that the properties of MTC aggregates with the peptides could not be totally interpreted by the binding through their secondary structures. The spatial arrangements of the amino acid residues also play an important role. This result is different from those of J-aggregates templated by well-ordered protein or longer polypeptides, indicating that the cyanine dye Jaggregates templated by peptides is a relatively complicated process. The detailed mechanism should be investigated further. Additionally, based on the strategy of peptide design in this paper, we could investigate mechanisms of the interaction between natural protein and small molecule assembly in more detail, and eventually grasp the essence and principle of the structure-function relationship of natural protein under the influence of small organic molecule. Supporting Information Available: Simulative pI titration curve of the small peptides and identification of cyanine dye MTC. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Pasternack, R. F.; Giannetto, A.; Pagano, P.; Gibbs, E. J. J. Am. Chem. Soc. 1991, 113, 7799. (b) Kim, O. K.; Je, J.; Jernigan, G.; Buckley, L.; Whitten, D. J. Am. Chem. Soc. 2006, 128, 510. (c) Balaban, T. S.; Bhise, A. D.; Fischer, M.; Linke-Schaetzel, M.; Roussel, C.; Vanthuyne, N. Angew.
J. Phys. Chem. B, Vol. 112, No. 29, 2008 8787 Chem., Int. Ed. 2003, 42, 2140. (d) Lohr, A.; Lysetska, M.; Wurthner, F. Angew. Chem., Int. Ed. 2005, 44, 5071. (2) Link, D. R.; Natale, G.; Shao, R.; Maclennan, J. E.; Clark, N. A.; Korblova, E.; Walba; D. M., Science 1997, 278, 1924. (3) James, T. H. The Theory of the Photographic Process; Macmillan Pub. Co.: New York, 1977. (4) Owen, D. J.; VanDerveer, D.; Schuster, G. B. J. Am. Chem. Soc. 1998, 120, 1705. (5) (a) Salvioli, S.; Ardizzoni, A.; Franceschi, C.; Cossarizza, A. FEBS Lett. 1997, 411, 77. (b) Higgins, D. A.; Kerimo, J.; VandenBout, D. A.; Barbara, P. F. J. Am. Chem. Soc. 1996, 118, 4049. (c) Tung, C. H.; Bredow, S.; Mahmood, U.; Weissleder, R. Bioconjugate Chem. 1999, 10, 892. (6) Li, N.; Yu, C. J.; Huang, F. Q. Nucleic Acids Res. 2005, 33, e37. (7) (a) Davila, J.; Harriman, A.; Gulliya, K. S. Photochem. Photobiol. 1991, 53, 1. (b) Diwu, Z. J.; Lown, J. W. Pharmacol. Ther. 1994, 63, 1. (c) Kudrevich, S.; Brasseur, N.; LaMadeleine, C.; Gilbert, S.; vanLier, J. E. J. Med. Chem. 1997, 40, 3897. (8) Furhop, J.-H.; Koenig, J. Membranes and molecular assemblies: the synkinetic approach; The Royal Society of Chemistry: Cambridge, 1994. (9) Herz, A. H. AdV. Colloid Interface Sci. 1977, 8, 237. (10) McRae, E. G.; Kasha, M. Physical Processes in Radiation Biology; Academic Press: New York, 1964. (11) (a) Honda, C.; Hada, H. Photogr. Sci. Eng. 1977, 21, 91. (b) Wang, M. M.; Silva, G. L.; Armitage, B. A. J. Am. Chem. Soc. 2000, 122, 9977. (12) Gibbs, E. J.; Jr., I. T.; Maestre, M. F.; Ellinas, P. A.; Pasternack, R. F. Biochem. Biophys. Res. Commun. 1988, 157, 350. (13) Cooper, T. M.; Stone, M. O. Langmuir 1998, 14, 6662. (14) Stryer, L.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 1411. (15) Nakashima, N.; Kunitake, T. J. Am. Chem. Soc. 1982, 104, 4261. (16) Seifert, J. L.; Connor, R. E.; Kushon, S. A.; Wang, M.; Armitage, B. A. J. Am. Chem. Soc. 1999, 121, 2987. (17) Zhang, Y.; Xiang, J.; Tang, Y.; Xu, G.; Yan, W. ChemPhysChem. 2007, 8, 224. (18) Hamer, F. M. The Chemistry of Heterocyclic Compounds; Interscience: New York, 1964; Vol. 18. (19) Ficken, G. E. The Chemistry of Synthetic Dyes; Academic Press: New York, 1971; Vol. 4. (20) Wang, J. M.; Wang, W.; Kollman, P. A.; Case, D. A. J. Mol. Graphics Modell. 2006, 25, 247. (21) Nakanishi, K.; Berova, N.; Woody, R. W. Circular Dichroism: Principles and Applications; VCH Publishers: New York, 1994. (22) Zhang, Y.; Du, H.; Tang, Y.; Xu, G.; Yan, W. Biophys. Chem. 2007, 128, 197.
JP803076D
