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Amphipathic cyclooctapeptides: interactions with detergent micelles and metal ions
Journal of Molecular Structure 733 (2005) 5–11 www.elsevier.com/locate/molstruc
Amphipathic cyclooctapeptides: interactions with detergent micelles and metal ions William Day Gates, Jack Rostas, Bobby Kakati, Maria Ngu-Schwemlein* Department of Chemistry, University of South Alabama, Mobile, AL 36688, USA Received 1 May 2004; revised 10 July 2004; accepted 15 July 2004 Available online 3 September 2004
Abstract In order to study the secondary structural preferences of amphipathic cyclopeptides in detergent assemblies and their interactions with metal ions, two basic amphiphatic cyclooctapeptides, c[(Lys-D-Lys)2-Xaa-D-Leu-Leu-D-Leu], where Xaa is Leu (P1) or Trp (P2), were studied by circular dichroism (CD) spectroscopy. In water, P1 exhibited an unordered secondary structure whereas P2 adopted a partial b-sheet structure. Temperature effect on the CD of these cyclopeptides showed small changes over the temperature range from 278 to 353 K. P1 showed low and non-specific binding affinity for metal ions (Ca2C, Zn2C, NaC, or LiC) in water whereas P2 did not exhibit any significant interaction with these ions. However, in the zwitterionic micellar detergent dodecylphosphocholine (DPC), P2 adopted a b-sheet structure, which exhibited a greater propensity for metal ion interactions (K1w103 MK1 and K2w102 MK1). Variable temperature CD studies on the peptide–metal ion complexes showed that these interactions are thermally reversible. Our results indicate that amphipathic cyclooctapeptides can co-assemble with micellar DPC and are capable of interacting with metal ions. This study will improve our ability to design a better metal ion sensing cyclopeptide in co-micellar assembles. q 2004 Elsevier B.V. All rights reserved. Keywords: Amphipathic cyclopeptides; Metal–ion interaction; b-sheet
1. Introduction Chemosensors for the detection of metal ions with high selectivity and specificity have wide applications in environmental chemistry and the medical sciences [1,2]. Although many types of molecules have been designed and synthesized as potential metal ion sensors, peptides and their derivatives, form an interesting class of sensors. Synthetic linear peptides have been designed for the detection of some metal ions [3–5] whereby the donor atoms reside within the peptide and/or non-natural amino acid residue, and ion binding modifies the emission properties of intrinsic or extrinsic fluorophores, which signals metal ion binding. Recently, it was reported that self-assembling of sensor components in surfactant aggregates is capable of * Corresponding author. Tel.: C1-251-460-7424; fax: C1-251-4607359. E-mail address: [email protected] (M. Ngu-Schwemlein). 0022-2860/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2004.07.031
complexing Cu2C, resulting in the quenching of entrapped dyes [6]. We are interested in extending this approach to incorporate amphiphatic (opposing faces of polarity) cyclopeptides, tagged with an intrinsic fluorophore, into inert detergent micelles to form a co-micellar assemblage. Cyclopeptides experience inherent conformational constraints, which enhance their selectivity in molecular interactions and recognition as opposed to that experienced by their relatively more flexible linear congeners. Additionally, cyclopeptides with opposing faces of polarity can coassemble with detergent micelles, which will improve solubilization of the cyclopeptide and its metal ion complex. Therefore, an optimized co-micellar assemblage of metal ion sensing cyclopeptides with detergent micelles may be an interesting approach towards the preparation of peptide based ion microsensors. Ghadiri et al. [7–9] have earlier reported that some hydrophobic cyclooctapeptides with alternating D-residues can form a stable ring-shaped structure, consisting of an anti-parallel b-sheet structure. Amphipathic analogues of
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these octapeptides are therefore suitable model cyclopeptides for studying the interactions of cyclopeptides with detergent micelles and metal ions. Understanding the structural changes of the cyclopeptides in co-micellar assemblies and in the presence of various metal ions is useful in working towards the design of fluorophore-tagged cyclopeptides as potential ion micro sensors. We have designed c[(Lys-D-Lys)2-Xaa-D-Leu-Leu-DLeu], where XaaZLeu (P1) or Trp (P2), in order to study the secondary structural preferences of amphipathic cyclopeptides in detergent assemblies by circular dichroism (CD), and their interactions with mono- and di- cationic metal ions such as Ca2C, Zn2C NaC or LiC. The structural preferences of these amphipathic peptides in water, micellar detergent, as well as their interactions with these metal ions are presented. Their metal ion binding affinity are assessed and compared. The preparation of P1 by convergent peptide synthesis is also described.
2. Materials and methods All chemicals were obtained from commercial suppliers and used without further purification. MALDI-TOF mass spectra were recorded on a Perseptive Biosystem MALDITOF mass spectrometer at Louisiana State University. The matrix compound was 2,5-dihydroxybenzoic acid (DHB). Crude cyclopeptides were purified on a Vydac C-4 reversed˚ , 5 mm, 10 mm! phase column (Vydac 214TP54, 300 A 250 mm). The mobile phase was H2O/0.1% trifluoroacetic acid (TFA) (A), and CH3 CN (50%)/isopropanol (50%)/0.1% TFA (B), delivered by the Rainin Dynamax HPLC system with UV monitoring at 214 nm. Cyclo(Lys-DLys)2-Trp-D-Leu-Leu-D-Leu (P2) was custom synthesized by SynPep, Inc. CD measurements were carried out on a Jasco 810 Spectrophotometer, equipped with a Peltier temperature controlled cell holder (PTC-423S/C). Spectra were recorded using 0.25–1 mg of peptide/ml solutions by sampling every 1 nm with an averaging time of 1 s, employing a 0.1 cm path length quartz cell. Each spectrum represents an average of four consecutive scans measured at 293 K, which was corrected by subtracting a corresponding blank solution. Stock dodecylphosphocholine (DPC) in 10% acetonitrile in water was prepared as 0.6 M solutions. Concentrated stock solutions of metal perchlorates (0.1, 1.0 or 5.0 M) were added to the peptide solutions to maintain various mole ratios of metal ion to peptide. The changes in molar ellipticity (deg MK1 cmK1 per residue) at specific wavelengths were determined prior to spectral smoothing using the Adjacent Averaging algorithm in the Microcal Origin 6.0 program (Microcal Software, Inc.). The metal ion–peptide binding constants were calculated from the extent of binding as previously reported by Blout [10]. The extent of binding, f, for a given amount of metal ion, c, added is f ðcÞZ ðMqc K Mqfree Þ=ðMqbound K Mqfree Þ; where Mq is the molar ellipticity per residue. The ion–peptide complexes
were assumed to be 1:1 followed by 2:1, based on the qualitative similarity of the CD spectra following metal ion titration, which showed isodichroic points indicating the existence of two state systems. The binding constant (K) for a metal ion and peptide interaction, PCM%PM (P for peptide and M for metal ion) is KZ[PM]/[P][M]Zf(c)/{1K f(c)}{M0Kf(c)P0} where M0 and P0 are the total concentrations of the metal ion and peptide, respectively [10]. 2.1. General method employed for the preparation of P1 Cyclo[(Lys{N3-2-Cl-CBZ}-D-Lys{N3-2-Cl-CBZ})2-(Leu(P1) was synthesized by solution phase peptide fragment coupling (Scheme 1). The linear tetrapeptides, Na-tBoc-(Lys{N3-2-Cl-CBZ}-D-Lys{N3-2-Cl-CBZ})2-OH and (Leu-D-Leu)2-Oa-phenacyl ester, were prepared by using the Na-t-Boc/Ca-O-phenacyl ester protecting group combination as previously reported for similar peptides [11,12]. Amino acid and peptide [2C2] couplings were mediated by 1hydroxy-7-azabenzotriazole (HOAT)/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)/N-methylmorpholine (NMM) in dichloromethane following the procedure reported earlier. Head-to-tail cyclization was conducted under dilution using benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP)/diisopropylethylamine (DIPEA) in dimethyformamide (DMF). The side chain 2-ClCBZ protecting groups were removed by catalytic hydrogenation over 10% Pd/C. The characterization of some of these hydrophobic peptides by optimized MALDI MS techniques
D-Leu)2]
Scheme 1. Synthesis of P1 by the fragment coupling approach.
W.D. Gates et al. / Journal of Molecular Structure 733 (2005) 5–11
has been described [13]. The purity of the intermediate peptides was checked by reversed phase HPLC analysis.
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Na-t-Boc-(Lys{N3-2-Cl-CBZ}-D-Lys{N3-2-Cl-CBZ})2OH (1.0 mmol) and (Leu- D -Leu) 2-O a-phenacyl ester (1.0 mmol) were dissolved in DMF (20 ml). Collidine (2.2 mmol) and N-{(dimethylamino)-HK1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene}-N-methylmethan-aminiumhexafluoro-phosphate N-oxide (HATU) were added, and the reaction mixture was stirred for 18 h at 298 K. The solvent was removed under high vacuum rotary evaporation and the residue was dissolved in ethylacetate (100 ml), and extracted with 1 M HCl (2!100 ml), 0.5 M NaHCO3(2!100 ml) and saturated NaCl (50 ml). Evaporation of solvents gave the octapeptide, t-Boc-(Lys{N3-2-ClCBZ}-D-Lys{N3-2-Cl-CBZ})2-(Leu-D-Leu)2-O-phenacyl ester. C-terminal deprotection with Zn in AcOH, followed by deprotection with TFA/CH2Cl2 (7:3) gave TFA$(Lys{N3-2-Cl-CBZ}-D-Lys{N3-2-Cl-CBZ})2-(Leu-DLeu)2-OH following precipitation from cold ether (85%, over 3 steps). MALDI-MS [MCH]C/1655 (calculated 1657), [MCNa]C/1677 (calculated 1679), and [MC K]C/1693 (calculated 1695).
and HATU, respectively. Optimization of the [4C4] coupling gave the octapeptides in purified yields ranging from 72 to 85%, with negligible racemization as detected by Marfey’s test [14]. The yields for purified linear peptides, from peptide couplings and deprotections, ranged from 85% to almost quantitative yields. Head-to-tail cyclization of the octapeptide to form the cyclooctapeptide was conducted under dilute conditions to avoid cyclodimerization and oligomerization [15]. This cyclization was also conducted under various conditions with some common peptide coupling activators. Our experimental conditions yielded optimal cyclization with PyBOP. Although peptide cyclization has been reported to be facilitated by C-terminal D-amino acid residues, this reaction turned out to be the yield-limiting step in the preparation of P1. The characterization of some of the intermediate hydrophobic peptides, containing the acid labile t-Boc-protecting group, by MALDI-TOF mass spectrometry were conducted by using an optimized procedure as reported earlier [13]. Accordingly, most of the hydrophobic peptides were detected as cation-adducted pseudomolecular ions due to the lack of suitable protonation sites. The amphipathic P1 was detected as protonated molecular ions and sodiated pseudomolecular ions, which readily exchanged with added AgC to form silver-adducted peudomolecular ions.
2.3. Cyclization of the octapeptide
3.2. CD studies
A solution of TFA (Lys{N3-2-Cl-CBZ}-D-Lys{N3-2-ClCBZ}) 2 -(Leu- D -Leu) 2-OH (0.11 mmol), PyBOP (0.16 mmol) in anhydrous DMF (225 ml) was stirred at 298 K. DIPEA (0.42 mmol) in 10 ml DMF was added dropwise over 1 h. After a further 5 h of stirring, the solution was concentrated under reduced pressure. The residue was triturated in dichloromethane (30 ml) and cold ether (45 ml) and the precipitate was isolated by centrifugation at 1200 rpm. Yield, 57%. MALDI-MS [MCNa]C/1664 (calculated 1661). The hydrophobic cyclopeptide (0.02 mmol) was dissolved in 90% glacial acetic acid (50 ml) and hydrogenated over 10% Pd/C for 18 h. After removal of Pd/C over Celite, the solvents were evaporated under reduced pressure. The crude product (88%) was purified on a reversed phase C-4 column to give the trifluoroacetate salt of the peptide as a white fluffy product following lyophilization. Conditions were 20% B to 45% B over 30 min with a flow rate of 5 ml/min. RP-HPLC trZ 15 min. MALDI-MS [MCH]C/966 (calculated 966) and [MCNa]C/988 (calculated 988).
3.2.1. Temperature effect The CD of a polypeptide is derived from the spectroscopic interactions amongst its amide chromophore. Accordingly, the cyclopeptide that adopts a specific peptide backbone-folding pattern (secondary structure) will show a characteristic CD spectrum. The CD spectrum of P1 shows a negative CD band at ca. 195 nm, a small positive band at 215 nm, and a small negative shoulder near 230 nm, which is characteristic of unordered secondary peptide structure [16] (Fig. 1A). P2 in H2O gave a CD with two negative bands at 202 and 223 nm, which indicates a partial b-sheet structure (Fig. 1B). The CD spectra of these heterochiral peptides show a marked negative pp* band at 195 nm (P1) and 202 nm (P2), and a weaker negative np* band at 230 nm (P1) and 223 nm (P2) [16,17]. In order to analyze the conformational flexibility of these peptide solution structures, a CD study involving temperature dependency was conducted. Both peptides showed small CD/temperature effect, accompanied by a decrease in molar ellipticity of the pp* band, which is also slightly red shifted at higher temperatures, with common isodichroic points at ca. 202 nm for P1 and ca. 194 nm for P2.
2.2. Linear octapeptide preparation
3. Results and discussion 3.1. Synthesis The linear precursor of P1 was successfully prepared by [2C2] followed by [4C4] fragment couplings with HOAT
3.2.2. Titrations with metal ions and DPC The addition of Ca2C to P1 produced changes in the CD spectrum. The weak negative band at 230 nm increased in intensity and shifted to 221 nm whereas the negative band at 195 nm gradually decreased and then increased in intensity
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Fig. 2. CD spectral change of P1 in H2O following titration with Ca2C at Ca2C/P1 ratios from 0 to 200 at 25 increments, as indicated by arrows.
concentrations of a neutral zwitterionic detergent, DPC, was conducted (Fig. 3). At low DPC concentrations, the CD of P2 does not change significantly. However, a conformational change from partial b-sheet to one with predominantly b-sheet structure occurs at the critical micelle concentration of DPC (ca. 8 mM). This indicates that P2 does not interact with the individual detergent molecules but it undergoes a structural transition in the presence of detergent micelles. In contrast, the unordered peptide, P1, did not undergo this structural transition in micelles.
Fig. 1. Variable temperature CD of P1 (A), and P2 (B) in H2O, from 278 to 353 K at 15 K increments as indicated by arrows.
to form a positive band at ca. 195 upon stepwise addition of Ca2C. The CD data indicates that the unordered cyclopeptide, P1, undergoes a secondary structural transition to a characteristic b-sheet structure in the presence of Ca2C (Fig. 2). The np* and pp* maxima are of approximately equal magnitude, which is associated with a low degree of twist in the b-sheet structure as reported for polypeptide models such as poly(Leu-Lys) [18,19]. Similar CD data were obtained when P1 was titrated with Zn2C, LiC, or NaC. These changes in CD, from an unordered peptide backbone structure to a b-sheet structure upon addition of metal ions, are probably due to complex formation involving the peptide bonds, which resulted in the blue shift of the np* transition. The CD of the complexes can also be correlated with the anti-parallel b-sheet and ‘ringlike’ conformation of the cyclooctapeptides as reported by Ghadiri et al. [7,9]. Although P2 adopts a partial b-sheet structure in water, it did not undergo a secondary structural transition in the presence of Ca2C, Zn2C, LiC, or NaC. In order to investigate the interactions of these amphipathic cyclopeptides with detergent molecules, the CD of P2 in varying
3.2.3. Titrations of cyclopeptide and detergent co-micelles with metal ions The metal ion binding properties of P1 and P2 with Ca2C, Zn2C, LiC and NaC in micellar detergent(s) were then examined. Each perchlorate salt of the metal was titrated separately into solutions containing the peptide and detergent co-micelles, and the CD spectrum was recorded following each titration. CD data showed that the unordered
Fig. 3. CD spectra of P2 in H2O (—) containing DPC, 1 mM (---), 2 mM ($$$$), 4 mM (-$-$), 6 mM (-$$-), 8 mM (- -), and 10 mM ($$$$).
W.D. Gates et al. / Journal of Molecular Structure 733 (2005) 5–11
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Fig. 4. CD spectra of P1 in 10 mM DPC following titration with Ca2C (—), Zn2C ($$$$) and NaC (---) at metal ion/P1 ratios of 300.
peptide, P1, in 10 mM DPC gradually adopted a twisted beta-sheet structure in the presence of excess Ca2C or Zn2C (Fig. 4). In the presence of NaC, it maintained a lower degree of twisting in the b-sheet structure. However, its interaction with these metal ions remained weak and non-specific. In contrast, P2 exhibited relatively stronger interactions with these metals. The CD data of P2 in 10 mM DPC or at higher DPC concentrations, following titration with Ca2C, showed an increase in molar ellipticity in both the pp* and np* bands, with isodichroic points at ca. 196 and 214 nm (Fig. 5A). The latter indicates the presences of a two-state system, which could correlate to the free peptide and metal ion bound peptide. The pp* band at 202 nm is stronger than the np* band at 226 nm. This shows that P2 forms a slightly twisted b-sheet structure(s) as the Ca2C to peptide ratio increases to five. The relative changes in molar ellipticity at 205 nm following titrations with Ca2C, Zn2C, NaC or LiC show no significant differences among the first binding constants for these ions (Fig. 6A). At increasing Ca2C to peptide mole ratios from six to sixty, the CD data of P2 showed a decrease in the molar ellipticity in both the pp* and np* bands, which are also blue shifted. These spectral changes are probably due to complex formation involving the peptide bonds. The presence of isodichroic points at ca. 198 and 218 nm (Fig. 5B) also shows that a two-state system exists. The relative changes in molar ellipticity at 230 nm following titrations with Ca2C, Zn2C, NaC and LiC shows a hyperbolic curve (Fig. 6B). In order to assess the ability of the cyclopeptides to bind these metal ions, binding constants were calculated from the extend of binding, which was determined from the CD data by titrations of a fixed concentration of the peptide with varying concentrations of the metal ion, as previously described by Blout [10]. The structurally unordered P1 showed a weak and non-specific interaction for Ca2C,
Fig. 5. CD spectral change of P2 in 10 mM DPC following titration with Ca2C at Ca2C/P2 ratios from 0 to 5 (A), and from 6 to 60 (B), as indicated by arrows.
Zn2C, NaC and LiC, with a low binding constant of ca. 8! 101 MK1 for Ca2C (Fig. 2). Although P2 adopted a partial b-sheet structure in H2O, it did not show any significant interactions with these metal ions. However, in DPC micelles, P2 adopted a b-sheet structure, which had a greater propensity to interact with metal ions. Its average binding constants for various metal ions are Ca2C [4! 103 MK1 (K1), 6!102 MK1 (K2)], Zn2C [2!103 MK1 (K1), 4!102 MK1 (K2)], and Na2C and LiC (1.5!103 MK1 (K1), 1!102 MK1 (K2)] (Fig. 6). 3.2.4. Reversibility of interactions with metal ions In order to obtain information about the stability of the metal ion(s)–peptide complexes in 10 mM DPC, a temperature CD was conducted. Fig. 7A shows the CD spectra of the Ca2C–peptide P1 complex in 10 mM DPC following equilibration at 293 K, 363 K, and subsequent cooling to 293 K. When the Ca2C–peptide P1 complex was heated to 363 K, the CD of the equilibrated solution became similar to the CD of the free peptide P1 at 363 K (Fig. 1A), which
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Fig. 6. Change in molar ellipticity at 205 nm (A) and 230 nm (B) following titrations with various mole ratios of Ca2C (&), Zn2C (C), NaC (:), and LiC (;) to P2.
indicated the dissociation of the ion bound peptide. On cooling this solution back to 293 K, the CD data showed that the ion–peptide complex was reconstituted, indicating that the ion–peptide complex is thermally reversible. Similar results were observed for the metal ion(s)– peptide P2 complexes in 10 mM DPC. Fig. 7B shows the thermal reversibility of the complex formation. Although the Ca2C–P2 complex dissociated at 363 K, P2 retained its association with the DPC micelles.
4. Conclusion The purpose of this work is to study the interactions of two model amphiphatic cyclopeptides with detergent micelles and some common metal ions in order to facilitate the design of cyclopeptides as sensors for metal ions. The above results indicate that amphiphatic cyclopeptides, P1 and P2, undergo conformational changes and adopt
Fig. 7. CD spectra of P1 in 10 mM DPC and 120 mol ratio of Ca2C (A), and P2 in 10 mM DPC and 50 mol ratio of Ca2C (B), at 293 K (—), 363 K (----), cooling back to 293 K ($$$$), and at 293 K (-$$-) without Ca2C.
a b-sheet structure in the presence of metal ions, such as Ca2C, Zn2C, NaC and LiC, or when associated with DPC micelles. P2, which contained a Trp residue, exhibited a greater propensity to interact with metal ions in detergent micelles. These metal ion induced structural changes will play an important role in signaling the presence of metal ions by modulating the fluorescence of an intrinsic fluorophore. Although these model amphipathic cyclooctapeptides exhibit relatively smaller binding constants for these metal ions when compared to the ionophoric depsipeptide, Valinomycin (K ca. 5!105 MK1 for KC) [20], the cyclooctapeptide scaffold could be chemically modified to optimize its metal ion interaction. For example, analogues of P2 with selective thionated peptide bonds could increase its association with heavy metal ions. Additionally, the substitution of the basic Lys residues in P2 with acidic carboxylic groups or phosphates could enhance electrostatic interactions with cations. Fluorescence studies on P2 will be reported later. These studies will be useful in designing derivatives of amphiphatic
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cyclopeptides, with optimal metal ion binding moieties, and a selective fluorophore for specific signal transduction.
Acknowledgements The National Science Foundation, grant #CHE 0031097, supported this work. We also wish to express our appreciation to Kari Green-Church and Steven Macha of Louisiana State University for the MALDI TOF mass spectra, and we acknowledge helpful discussions with Dr Mark McLaughlin of University of South Florida.
References [1] C. Bargossi, M.C. Fiorini, M. Montalti, L. Prodi, N. Zaccheroni, Coord. Chem. Rev. 208 (2000) 17–32. [2] A.W. Czarnik, Supramolecular chemistry fluorescence, and sensing, in: A.W. Czarnik (Ed.),, Fluorescent Chemosensors for Ion and Molecular Recognition, ACS Symposium Series 538, ACS, Washinghton, DC, 1993, Chapter 1, and references therein. [3] (a) A. Torrado, G.K. Walkup, B. Imperiali, J. Am. Chem. Soc. 120 (1998) 609–610; (b) M.D. Shults, D.A. Pearce, B. Imperiali, J. Am. Chem. Soc. 125 (2003) 10591–10597. [4] Y. Yang, X. Cao, J. Orbulescu, V. Konka, F.M. Andreopoulos, S.M. Pham, R.M. Leblanc, Anal. Chem. 75 (2003) 1706–1712. [5] S. Bhattacharya, M. Thomas, Tetrahedron Lett. 41 (2000) 10313– 10317.
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[6] M. Berton, F. Mancin, G. Stoccherro, P. Tecilla, U. Tonellato, Langmuir 17 (2001) 7521. [7] H. Rapaport, H.S. Kim, K. Kjaer, P.B. Howes, S. Cohen, S.J. Als-Nielsen, M.R. Ghadiri, L. Leiserowitz, M. Lahav, J. Am. Chem. Soc. 121 (1999) 1186–1191. [8] J.D. Hartgerink, T.D. Clark, M.R. Ghadiri, Chem. Eur. J. 4 (1998) 1367–1372. [9] M.R. Ghadiri, J.R. Granja, R.A. Milligan, D.E. McRee, N. Khazanovich, Nature 366 (1993) 324–327. [10] V. Madison, M. Atreyi, C.M. Deber, E.R. Blout, J. Am. Chem. Soc. 96 (1974) 6725–6734. [11] M. Ngu-Schwemlein, Z. Zhou, T. Bowie, R. Eden, J. Mol. Struct. 655 (2003) 58–68. [12] X. Wang, W. Daly, P. Russo, M. Ngu-Schwemlein, Biomacromolecules 2 (4) (2001) 1214–1219. [13] K.B. Green-Church, P.A. Limbach, Matrix-assisted laser desorption/ ionization mass spectrometry of hydrophobic peptides, Anal. Chem. 70 (1998) 5322–5325. [14] C. B’Hymer, M. Montes-Bayon, J.A. Caruso, J. Sep. Sci. 26 (2003) 7–19. [15] M. El Haddadi, F. Cavelier, E. Vives, A. Azmani, J. Verducci, J. Martinez, J. Pept. Sci. 6 (2000) 560–570. [16] N. Sreerama, R.W. Woody, Circular dichroism of peptides and proteins in: N. Berova, K. Nakanishi, R.W. Woody (Eds.), Circular Dichroism, Principles and Applications, second ed., Wiley, New York, 2000, pp. 601–620. [17] S.M. Kelly, N.C. Price, Biochim. Biophys. Acta 1338 (1997) 161–185. [18] S. Brahms, J. Brahms, G. Spach, A. Brack, Proc. Natl Acad. Sci. USA 74 (1977) 3208–3212. [19] R.W. Woody, Theory of circular dichroism of proteins in: G.D. Fasman (Ed.),, Circular Dichroism and the Conformational Analysis of Biomolecules, Plenum Press, New York, 1996, pp. 25–68. [20] V. Vodyanoy, Langmuir 10 (1994) 1354–1357.
Amphipathic cyclooctapeptides: interactions with detergent micelles and metal ions William Day Gates, Jack Rostas, Bobby Kakati, Maria Ngu-Schwemlein* Department of Chemistry, University of South Alabama, Mobile, AL 36688, USA Received 1 May 2004; revised 10 July 2004; accepted 15 July 2004 Available online 3 September 2004
Abstract In order to study the secondary structural preferences of amphipathic cyclopeptides in detergent assemblies and their interactions with metal ions, two basic amphiphatic cyclooctapeptides, c[(Lys-D-Lys)2-Xaa-D-Leu-Leu-D-Leu], where Xaa is Leu (P1) or Trp (P2), were studied by circular dichroism (CD) spectroscopy. In water, P1 exhibited an unordered secondary structure whereas P2 adopted a partial b-sheet structure. Temperature effect on the CD of these cyclopeptides showed small changes over the temperature range from 278 to 353 K. P1 showed low and non-specific binding affinity for metal ions (Ca2C, Zn2C, NaC, or LiC) in water whereas P2 did not exhibit any significant interaction with these ions. However, in the zwitterionic micellar detergent dodecylphosphocholine (DPC), P2 adopted a b-sheet structure, which exhibited a greater propensity for metal ion interactions (K1w103 MK1 and K2w102 MK1). Variable temperature CD studies on the peptide–metal ion complexes showed that these interactions are thermally reversible. Our results indicate that amphipathic cyclooctapeptides can co-assemble with micellar DPC and are capable of interacting with metal ions. This study will improve our ability to design a better metal ion sensing cyclopeptide in co-micellar assembles. q 2004 Elsevier B.V. All rights reserved. Keywords: Amphipathic cyclopeptides; Metal–ion interaction; b-sheet
1. Introduction Chemosensors for the detection of metal ions with high selectivity and specificity have wide applications in environmental chemistry and the medical sciences [1,2]. Although many types of molecules have been designed and synthesized as potential metal ion sensors, peptides and their derivatives, form an interesting class of sensors. Synthetic linear peptides have been designed for the detection of some metal ions [3–5] whereby the donor atoms reside within the peptide and/or non-natural amino acid residue, and ion binding modifies the emission properties of intrinsic or extrinsic fluorophores, which signals metal ion binding. Recently, it was reported that self-assembling of sensor components in surfactant aggregates is capable of * Corresponding author. Tel.: C1-251-460-7424; fax: C1-251-4607359. E-mail address: [email protected] (M. Ngu-Schwemlein). 0022-2860/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2004.07.031
complexing Cu2C, resulting in the quenching of entrapped dyes [6]. We are interested in extending this approach to incorporate amphiphatic (opposing faces of polarity) cyclopeptides, tagged with an intrinsic fluorophore, into inert detergent micelles to form a co-micellar assemblage. Cyclopeptides experience inherent conformational constraints, which enhance their selectivity in molecular interactions and recognition as opposed to that experienced by their relatively more flexible linear congeners. Additionally, cyclopeptides with opposing faces of polarity can coassemble with detergent micelles, which will improve solubilization of the cyclopeptide and its metal ion complex. Therefore, an optimized co-micellar assemblage of metal ion sensing cyclopeptides with detergent micelles may be an interesting approach towards the preparation of peptide based ion microsensors. Ghadiri et al. [7–9] have earlier reported that some hydrophobic cyclooctapeptides with alternating D-residues can form a stable ring-shaped structure, consisting of an anti-parallel b-sheet structure. Amphipathic analogues of
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these octapeptides are therefore suitable model cyclopeptides for studying the interactions of cyclopeptides with detergent micelles and metal ions. Understanding the structural changes of the cyclopeptides in co-micellar assemblies and in the presence of various metal ions is useful in working towards the design of fluorophore-tagged cyclopeptides as potential ion micro sensors. We have designed c[(Lys-D-Lys)2-Xaa-D-Leu-Leu-DLeu], where XaaZLeu (P1) or Trp (P2), in order to study the secondary structural preferences of amphipathic cyclopeptides in detergent assemblies by circular dichroism (CD), and their interactions with mono- and di- cationic metal ions such as Ca2C, Zn2C NaC or LiC. The structural preferences of these amphipathic peptides in water, micellar detergent, as well as their interactions with these metal ions are presented. Their metal ion binding affinity are assessed and compared. The preparation of P1 by convergent peptide synthesis is also described.
2. Materials and methods All chemicals were obtained from commercial suppliers and used without further purification. MALDI-TOF mass spectra were recorded on a Perseptive Biosystem MALDITOF mass spectrometer at Louisiana State University. The matrix compound was 2,5-dihydroxybenzoic acid (DHB). Crude cyclopeptides were purified on a Vydac C-4 reversed˚ , 5 mm, 10 mm! phase column (Vydac 214TP54, 300 A 250 mm). The mobile phase was H2O/0.1% trifluoroacetic acid (TFA) (A), and CH3 CN (50%)/isopropanol (50%)/0.1% TFA (B), delivered by the Rainin Dynamax HPLC system with UV monitoring at 214 nm. Cyclo(Lys-DLys)2-Trp-D-Leu-Leu-D-Leu (P2) was custom synthesized by SynPep, Inc. CD measurements were carried out on a Jasco 810 Spectrophotometer, equipped with a Peltier temperature controlled cell holder (PTC-423S/C). Spectra were recorded using 0.25–1 mg of peptide/ml solutions by sampling every 1 nm with an averaging time of 1 s, employing a 0.1 cm path length quartz cell. Each spectrum represents an average of four consecutive scans measured at 293 K, which was corrected by subtracting a corresponding blank solution. Stock dodecylphosphocholine (DPC) in 10% acetonitrile in water was prepared as 0.6 M solutions. Concentrated stock solutions of metal perchlorates (0.1, 1.0 or 5.0 M) were added to the peptide solutions to maintain various mole ratios of metal ion to peptide. The changes in molar ellipticity (deg MK1 cmK1 per residue) at specific wavelengths were determined prior to spectral smoothing using the Adjacent Averaging algorithm in the Microcal Origin 6.0 program (Microcal Software, Inc.). The metal ion–peptide binding constants were calculated from the extent of binding as previously reported by Blout [10]. The extent of binding, f, for a given amount of metal ion, c, added is f ðcÞZ ðMqc K Mqfree Þ=ðMqbound K Mqfree Þ; where Mq is the molar ellipticity per residue. The ion–peptide complexes
were assumed to be 1:1 followed by 2:1, based on the qualitative similarity of the CD spectra following metal ion titration, which showed isodichroic points indicating the existence of two state systems. The binding constant (K) for a metal ion and peptide interaction, PCM%PM (P for peptide and M for metal ion) is KZ[PM]/[P][M]Zf(c)/{1K f(c)}{M0Kf(c)P0} where M0 and P0 are the total concentrations of the metal ion and peptide, respectively [10]. 2.1. General method employed for the preparation of P1 Cyclo[(Lys{N3-2-Cl-CBZ}-D-Lys{N3-2-Cl-CBZ})2-(Leu(P1) was synthesized by solution phase peptide fragment coupling (Scheme 1). The linear tetrapeptides, Na-tBoc-(Lys{N3-2-Cl-CBZ}-D-Lys{N3-2-Cl-CBZ})2-OH and (Leu-D-Leu)2-Oa-phenacyl ester, were prepared by using the Na-t-Boc/Ca-O-phenacyl ester protecting group combination as previously reported for similar peptides [11,12]. Amino acid and peptide [2C2] couplings were mediated by 1hydroxy-7-azabenzotriazole (HOAT)/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)/N-methylmorpholine (NMM) in dichloromethane following the procedure reported earlier. Head-to-tail cyclization was conducted under dilution using benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP)/diisopropylethylamine (DIPEA) in dimethyformamide (DMF). The side chain 2-ClCBZ protecting groups were removed by catalytic hydrogenation over 10% Pd/C. The characterization of some of these hydrophobic peptides by optimized MALDI MS techniques
D-Leu)2]
Scheme 1. Synthesis of P1 by the fragment coupling approach.
W.D. Gates et al. / Journal of Molecular Structure 733 (2005) 5–11
has been described [13]. The purity of the intermediate peptides was checked by reversed phase HPLC analysis.
7
Na-t-Boc-(Lys{N3-2-Cl-CBZ}-D-Lys{N3-2-Cl-CBZ})2OH (1.0 mmol) and (Leu- D -Leu) 2-O a-phenacyl ester (1.0 mmol) were dissolved in DMF (20 ml). Collidine (2.2 mmol) and N-{(dimethylamino)-HK1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene}-N-methylmethan-aminiumhexafluoro-phosphate N-oxide (HATU) were added, and the reaction mixture was stirred for 18 h at 298 K. The solvent was removed under high vacuum rotary evaporation and the residue was dissolved in ethylacetate (100 ml), and extracted with 1 M HCl (2!100 ml), 0.5 M NaHCO3(2!100 ml) and saturated NaCl (50 ml). Evaporation of solvents gave the octapeptide, t-Boc-(Lys{N3-2-ClCBZ}-D-Lys{N3-2-Cl-CBZ})2-(Leu-D-Leu)2-O-phenacyl ester. C-terminal deprotection with Zn in AcOH, followed by deprotection with TFA/CH2Cl2 (7:3) gave TFA$(Lys{N3-2-Cl-CBZ}-D-Lys{N3-2-Cl-CBZ})2-(Leu-DLeu)2-OH following precipitation from cold ether (85%, over 3 steps). MALDI-MS [MCH]C/1655 (calculated 1657), [MCNa]C/1677 (calculated 1679), and [MC K]C/1693 (calculated 1695).
and HATU, respectively. Optimization of the [4C4] coupling gave the octapeptides in purified yields ranging from 72 to 85%, with negligible racemization as detected by Marfey’s test [14]. The yields for purified linear peptides, from peptide couplings and deprotections, ranged from 85% to almost quantitative yields. Head-to-tail cyclization of the octapeptide to form the cyclooctapeptide was conducted under dilute conditions to avoid cyclodimerization and oligomerization [15]. This cyclization was also conducted under various conditions with some common peptide coupling activators. Our experimental conditions yielded optimal cyclization with PyBOP. Although peptide cyclization has been reported to be facilitated by C-terminal D-amino acid residues, this reaction turned out to be the yield-limiting step in the preparation of P1. The characterization of some of the intermediate hydrophobic peptides, containing the acid labile t-Boc-protecting group, by MALDI-TOF mass spectrometry were conducted by using an optimized procedure as reported earlier [13]. Accordingly, most of the hydrophobic peptides were detected as cation-adducted pseudomolecular ions due to the lack of suitable protonation sites. The amphipathic P1 was detected as protonated molecular ions and sodiated pseudomolecular ions, which readily exchanged with added AgC to form silver-adducted peudomolecular ions.
2.3. Cyclization of the octapeptide
3.2. CD studies
A solution of TFA (Lys{N3-2-Cl-CBZ}-D-Lys{N3-2-ClCBZ}) 2 -(Leu- D -Leu) 2-OH (0.11 mmol), PyBOP (0.16 mmol) in anhydrous DMF (225 ml) was stirred at 298 K. DIPEA (0.42 mmol) in 10 ml DMF was added dropwise over 1 h. After a further 5 h of stirring, the solution was concentrated under reduced pressure. The residue was triturated in dichloromethane (30 ml) and cold ether (45 ml) and the precipitate was isolated by centrifugation at 1200 rpm. Yield, 57%. MALDI-MS [MCNa]C/1664 (calculated 1661). The hydrophobic cyclopeptide (0.02 mmol) was dissolved in 90% glacial acetic acid (50 ml) and hydrogenated over 10% Pd/C for 18 h. After removal of Pd/C over Celite, the solvents were evaporated under reduced pressure. The crude product (88%) was purified on a reversed phase C-4 column to give the trifluoroacetate salt of the peptide as a white fluffy product following lyophilization. Conditions were 20% B to 45% B over 30 min with a flow rate of 5 ml/min. RP-HPLC trZ 15 min. MALDI-MS [MCH]C/966 (calculated 966) and [MCNa]C/988 (calculated 988).
3.2.1. Temperature effect The CD of a polypeptide is derived from the spectroscopic interactions amongst its amide chromophore. Accordingly, the cyclopeptide that adopts a specific peptide backbone-folding pattern (secondary structure) will show a characteristic CD spectrum. The CD spectrum of P1 shows a negative CD band at ca. 195 nm, a small positive band at 215 nm, and a small negative shoulder near 230 nm, which is characteristic of unordered secondary peptide structure [16] (Fig. 1A). P2 in H2O gave a CD with two negative bands at 202 and 223 nm, which indicates a partial b-sheet structure (Fig. 1B). The CD spectra of these heterochiral peptides show a marked negative pp* band at 195 nm (P1) and 202 nm (P2), and a weaker negative np* band at 230 nm (P1) and 223 nm (P2) [16,17]. In order to analyze the conformational flexibility of these peptide solution structures, a CD study involving temperature dependency was conducted. Both peptides showed small CD/temperature effect, accompanied by a decrease in molar ellipticity of the pp* band, which is also slightly red shifted at higher temperatures, with common isodichroic points at ca. 202 nm for P1 and ca. 194 nm for P2.
2.2. Linear octapeptide preparation
3. Results and discussion 3.1. Synthesis The linear precursor of P1 was successfully prepared by [2C2] followed by [4C4] fragment couplings with HOAT
3.2.2. Titrations with metal ions and DPC The addition of Ca2C to P1 produced changes in the CD spectrum. The weak negative band at 230 nm increased in intensity and shifted to 221 nm whereas the negative band at 195 nm gradually decreased and then increased in intensity
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W.D. Gates et al. / Journal of Molecular Structure 733 (2005) 5–11
Fig. 2. CD spectral change of P1 in H2O following titration with Ca2C at Ca2C/P1 ratios from 0 to 200 at 25 increments, as indicated by arrows.
concentrations of a neutral zwitterionic detergent, DPC, was conducted (Fig. 3). At low DPC concentrations, the CD of P2 does not change significantly. However, a conformational change from partial b-sheet to one with predominantly b-sheet structure occurs at the critical micelle concentration of DPC (ca. 8 mM). This indicates that P2 does not interact with the individual detergent molecules but it undergoes a structural transition in the presence of detergent micelles. In contrast, the unordered peptide, P1, did not undergo this structural transition in micelles.
Fig. 1. Variable temperature CD of P1 (A), and P2 (B) in H2O, from 278 to 353 K at 15 K increments as indicated by arrows.
to form a positive band at ca. 195 upon stepwise addition of Ca2C. The CD data indicates that the unordered cyclopeptide, P1, undergoes a secondary structural transition to a characteristic b-sheet structure in the presence of Ca2C (Fig. 2). The np* and pp* maxima are of approximately equal magnitude, which is associated with a low degree of twist in the b-sheet structure as reported for polypeptide models such as poly(Leu-Lys) [18,19]. Similar CD data were obtained when P1 was titrated with Zn2C, LiC, or NaC. These changes in CD, from an unordered peptide backbone structure to a b-sheet structure upon addition of metal ions, are probably due to complex formation involving the peptide bonds, which resulted in the blue shift of the np* transition. The CD of the complexes can also be correlated with the anti-parallel b-sheet and ‘ringlike’ conformation of the cyclooctapeptides as reported by Ghadiri et al. [7,9]. Although P2 adopts a partial b-sheet structure in water, it did not undergo a secondary structural transition in the presence of Ca2C, Zn2C, LiC, or NaC. In order to investigate the interactions of these amphipathic cyclopeptides with detergent molecules, the CD of P2 in varying
3.2.3. Titrations of cyclopeptide and detergent co-micelles with metal ions The metal ion binding properties of P1 and P2 with Ca2C, Zn2C, LiC and NaC in micellar detergent(s) were then examined. Each perchlorate salt of the metal was titrated separately into solutions containing the peptide and detergent co-micelles, and the CD spectrum was recorded following each titration. CD data showed that the unordered
Fig. 3. CD spectra of P2 in H2O (—) containing DPC, 1 mM (---), 2 mM ($$$$), 4 mM (-$-$), 6 mM (-$$-), 8 mM (- -), and 10 mM ($$$$).
W.D. Gates et al. / Journal of Molecular Structure 733 (2005) 5–11
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Fig. 4. CD spectra of P1 in 10 mM DPC following titration with Ca2C (—), Zn2C ($$$$) and NaC (---) at metal ion/P1 ratios of 300.
peptide, P1, in 10 mM DPC gradually adopted a twisted beta-sheet structure in the presence of excess Ca2C or Zn2C (Fig. 4). In the presence of NaC, it maintained a lower degree of twisting in the b-sheet structure. However, its interaction with these metal ions remained weak and non-specific. In contrast, P2 exhibited relatively stronger interactions with these metals. The CD data of P2 in 10 mM DPC or at higher DPC concentrations, following titration with Ca2C, showed an increase in molar ellipticity in both the pp* and np* bands, with isodichroic points at ca. 196 and 214 nm (Fig. 5A). The latter indicates the presences of a two-state system, which could correlate to the free peptide and metal ion bound peptide. The pp* band at 202 nm is stronger than the np* band at 226 nm. This shows that P2 forms a slightly twisted b-sheet structure(s) as the Ca2C to peptide ratio increases to five. The relative changes in molar ellipticity at 205 nm following titrations with Ca2C, Zn2C, NaC or LiC show no significant differences among the first binding constants for these ions (Fig. 6A). At increasing Ca2C to peptide mole ratios from six to sixty, the CD data of P2 showed a decrease in the molar ellipticity in both the pp* and np* bands, which are also blue shifted. These spectral changes are probably due to complex formation involving the peptide bonds. The presence of isodichroic points at ca. 198 and 218 nm (Fig. 5B) also shows that a two-state system exists. The relative changes in molar ellipticity at 230 nm following titrations with Ca2C, Zn2C, NaC and LiC shows a hyperbolic curve (Fig. 6B). In order to assess the ability of the cyclopeptides to bind these metal ions, binding constants were calculated from the extend of binding, which was determined from the CD data by titrations of a fixed concentration of the peptide with varying concentrations of the metal ion, as previously described by Blout [10]. The structurally unordered P1 showed a weak and non-specific interaction for Ca2C,
Fig. 5. CD spectral change of P2 in 10 mM DPC following titration with Ca2C at Ca2C/P2 ratios from 0 to 5 (A), and from 6 to 60 (B), as indicated by arrows.
Zn2C, NaC and LiC, with a low binding constant of ca. 8! 101 MK1 for Ca2C (Fig. 2). Although P2 adopted a partial b-sheet structure in H2O, it did not show any significant interactions with these metal ions. However, in DPC micelles, P2 adopted a b-sheet structure, which had a greater propensity to interact with metal ions. Its average binding constants for various metal ions are Ca2C [4! 103 MK1 (K1), 6!102 MK1 (K2)], Zn2C [2!103 MK1 (K1), 4!102 MK1 (K2)], and Na2C and LiC (1.5!103 MK1 (K1), 1!102 MK1 (K2)] (Fig. 6). 3.2.4. Reversibility of interactions with metal ions In order to obtain information about the stability of the metal ion(s)–peptide complexes in 10 mM DPC, a temperature CD was conducted. Fig. 7A shows the CD spectra of the Ca2C–peptide P1 complex in 10 mM DPC following equilibration at 293 K, 363 K, and subsequent cooling to 293 K. When the Ca2C–peptide P1 complex was heated to 363 K, the CD of the equilibrated solution became similar to the CD of the free peptide P1 at 363 K (Fig. 1A), which
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Fig. 6. Change in molar ellipticity at 205 nm (A) and 230 nm (B) following titrations with various mole ratios of Ca2C (&), Zn2C (C), NaC (:), and LiC (;) to P2.
indicated the dissociation of the ion bound peptide. On cooling this solution back to 293 K, the CD data showed that the ion–peptide complex was reconstituted, indicating that the ion–peptide complex is thermally reversible. Similar results were observed for the metal ion(s)– peptide P2 complexes in 10 mM DPC. Fig. 7B shows the thermal reversibility of the complex formation. Although the Ca2C–P2 complex dissociated at 363 K, P2 retained its association with the DPC micelles.
4. Conclusion The purpose of this work is to study the interactions of two model amphiphatic cyclopeptides with detergent micelles and some common metal ions in order to facilitate the design of cyclopeptides as sensors for metal ions. The above results indicate that amphiphatic cyclopeptides, P1 and P2, undergo conformational changes and adopt
Fig. 7. CD spectra of P1 in 10 mM DPC and 120 mol ratio of Ca2C (A), and P2 in 10 mM DPC and 50 mol ratio of Ca2C (B), at 293 K (—), 363 K (----), cooling back to 293 K ($$$$), and at 293 K (-$$-) without Ca2C.
a b-sheet structure in the presence of metal ions, such as Ca2C, Zn2C, NaC and LiC, or when associated with DPC micelles. P2, which contained a Trp residue, exhibited a greater propensity to interact with metal ions in detergent micelles. These metal ion induced structural changes will play an important role in signaling the presence of metal ions by modulating the fluorescence of an intrinsic fluorophore. Although these model amphipathic cyclooctapeptides exhibit relatively smaller binding constants for these metal ions when compared to the ionophoric depsipeptide, Valinomycin (K ca. 5!105 MK1 for KC) [20], the cyclooctapeptide scaffold could be chemically modified to optimize its metal ion interaction. For example, analogues of P2 with selective thionated peptide bonds could increase its association with heavy metal ions. Additionally, the substitution of the basic Lys residues in P2 with acidic carboxylic groups or phosphates could enhance electrostatic interactions with cations. Fluorescence studies on P2 will be reported later. These studies will be useful in designing derivatives of amphiphatic
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cyclopeptides, with optimal metal ion binding moieties, and a selective fluorophore for specific signal transduction.
Acknowledgements The National Science Foundation, grant #CHE 0031097, supported this work. We also wish to express our appreciation to Kari Green-Church and Steven Macha of Louisiana State University for the MALDI TOF mass spectra, and we acknowledge helpful discussions with Dr Mark McLaughlin of University of South Florida.
References [1] C. Bargossi, M.C. Fiorini, M. Montalti, L. Prodi, N. Zaccheroni, Coord. Chem. Rev. 208 (2000) 17–32. [2] A.W. Czarnik, Supramolecular chemistry fluorescence, and sensing, in: A.W. Czarnik (Ed.),, Fluorescent Chemosensors for Ion and Molecular Recognition, ACS Symposium Series 538, ACS, Washinghton, DC, 1993, Chapter 1, and references therein. [3] (a) A. Torrado, G.K. Walkup, B. Imperiali, J. Am. Chem. Soc. 120 (1998) 609–610; (b) M.D. Shults, D.A. Pearce, B. Imperiali, J. Am. Chem. Soc. 125 (2003) 10591–10597. [4] Y. Yang, X. Cao, J. Orbulescu, V. Konka, F.M. Andreopoulos, S.M. Pham, R.M. Leblanc, Anal. Chem. 75 (2003) 1706–1712. [5] S. Bhattacharya, M. Thomas, Tetrahedron Lett. 41 (2000) 10313– 10317.
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[6] M. Berton, F. Mancin, G. Stoccherro, P. Tecilla, U. Tonellato, Langmuir 17 (2001) 7521. [7] H. Rapaport, H.S. Kim, K. Kjaer, P.B. Howes, S. Cohen, S.J. Als-Nielsen, M.R. Ghadiri, L. Leiserowitz, M. Lahav, J. Am. Chem. Soc. 121 (1999) 1186–1191. [8] J.D. Hartgerink, T.D. Clark, M.R. Ghadiri, Chem. Eur. J. 4 (1998) 1367–1372. [9] M.R. Ghadiri, J.R. Granja, R.A. Milligan, D.E. McRee, N. Khazanovich, Nature 366 (1993) 324–327. [10] V. Madison, M. Atreyi, C.M. Deber, E.R. Blout, J. Am. Chem. Soc. 96 (1974) 6725–6734. [11] M. Ngu-Schwemlein, Z. Zhou, T. Bowie, R. Eden, J. Mol. Struct. 655 (2003) 58–68. [12] X. Wang, W. Daly, P. Russo, M. Ngu-Schwemlein, Biomacromolecules 2 (4) (2001) 1214–1219. [13] K.B. Green-Church, P.A. Limbach, Matrix-assisted laser desorption/ ionization mass spectrometry of hydrophobic peptides, Anal. Chem. 70 (1998) 5322–5325. [14] C. B’Hymer, M. Montes-Bayon, J.A. Caruso, J. Sep. Sci. 26 (2003) 7–19. [15] M. El Haddadi, F. Cavelier, E. Vives, A. Azmani, J. Verducci, J. Martinez, J. Pept. Sci. 6 (2000) 560–570. [16] N. Sreerama, R.W. Woody, Circular dichroism of peptides and proteins in: N. Berova, K. Nakanishi, R.W. Woody (Eds.), Circular Dichroism, Principles and Applications, second ed., Wiley, New York, 2000, pp. 601–620. [17] S.M. Kelly, N.C. Price, Biochim. Biophys. Acta 1338 (1997) 161–185. [18] S. Brahms, J. Brahms, G. Spach, A. Brack, Proc. Natl Acad. Sci. USA 74 (1977) 3208–3212. [19] R.W. Woody, Theory of circular dichroism of proteins in: G.D. Fasman (Ed.),, Circular Dichroism and the Conformational Analysis of Biomolecules, Plenum Press, New York, 1996, pp. 25–68. [20] V. Vodyanoy, Langmuir 10 (1994) 1354–1357.