Langmuir 1999, 15, 5467-5473
5467
Structural Transition of Nonionic Peptide Aggregates in Aqueous Medium M. Murugesan, M. Aulice Scibioh, and R. Jayakumar* Bioorganic Laboratory, Central Leather Research Institute, Adyar, Madras 600 020, India Received October 7, 1998. In Final Form: April 22, 1999 The structural transition of a nonionic peptide aggregate Boc-Leu-Asn-OEt (1) in aqueous medium was studied by 1H NMR, UV, and fluorescence spectroscopic techniques. Fluorescence studies of pyrene in the peptide aggregate indicate a considerable change in the interior dielectric constant above the transition temperature 40 °C (Tm), while fluorescence studies of ANS reveal significant changes in micellar microviscosity on structural transition. ANS binding studies were also carried out to study the nature of interaction in the two types of aggregates. The results suggest that the binding of ANS at lower temperature (40 °C) the interaction is enthalpic in nature. The temperature coefficients of amide proton chemical shifts of the peptide indicate that the hydrogen bonding pattern changes during the structural transition. The results indicate that the structural transition is due to the melting of “solid-like” aggregates to the “molten state” of the peptide in aqueous medium. This study has been extended to other nonionic peptides such as Boc-Ala-Asn-OEt (2) and Boc-(Ile)2NH(CH2CH2O)3CH3 (3). Aggregation studies of peptides 2 and 3 have been carried out, and their thermodynamic parameters such as ∆G°m, ∆H°m, ∆S°m, and ∆C°p are determined from the critical micellar concentration of the respective peptides. Here we present the comparative studies on the aggregate-forming characteristics and structural transition of peptides 1, 2 and 3.
Introduction Peptide aggregates provide useful structural and functional models to study complex bioaggregates.1 Possible analogies between peptide self-assembly and protein folding have also been suggested.2 Peptide aggregates take up different aggregative pathways such as spherical micelles,2 membranes,3 and nanotubules4 depending upon the structure of individual surfactant monomers and solvent conditions. From the thermodynamic studies of aggregation of peptides, we have concluded2 that these systems change their structure abruptly from one to another at a certain temperature. Furthermore, with respect to the monomeric conformational preference, we have suggested that the break point on the structural parameters against temperature is caused by a structural transition of the aggregate.2c However, this suggestion remains unexplored in the light of thermodynamic information. The present study emphasizes the thermodynamic interpretation in structural transition. Fluorescence probes such as pyrene and 8-anilino-1-naphthalene sulfonic acid (ANS) were used to characterize the polarity and microviscosity of the aggregate interior in different structural forms. 1H NMR studies were carried out to get information about the nature of hydrogen bonding pattern above and below the phase transition temperature (Tm). Experimental Section The synthesis and purification of the peptide Boc-Leu-AsnOEt (1) have been described elsewhere.5 The peptide Boc-Ala(1) (a) Shimizu, T.; Tanaka, Y.; Tsuda, K. Bull. Chem. Soc. Jpn. 1985, 58, 3436. (b) Shimizu, T.; Mori, M.; Minamikawa, H.; Hato, M. J. Chem. Soc., Chem. Commun. 1990, 183. (2) (a) Jayakumar, R.; Mandal, A. B.; Manoharan, P. T. J. Chem. Soc. Chem. Commun. 1993, 853. (b) Jayakumar, R.; Dhathathreyan, A.; Ramasami, T. Indian J. Chem. 1993, 32A, 373. (c) Jayakumar, R.; Jayanthi, C.; Gomathy, L. Int. J. Pep. Prot. Res. 1995, 45, 129. (d) Mandal, A. B.; Jayakumar, R. J. Chem. Soc., Faraday Trans. 1994, 90, 161. (3) Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3334. (4) Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.; Khazanovich, N. Nature 1993, 399, 324.
Asn-OCH2CH3 (2) was prepared by the solution phase method using the dicyclohexylcarbodiimide hydroxysuccinimide technique.6 tert-Butyloxycarbonyl alanyl N-hydroxysuccinimide ester (2.86 g, 10 mM), L-asparaginylethyl ester hydrochloride (1.91 g, 10 mM), and triethylamine (1.4 mL, 10 mM) were added to dry dioxane (33 mL); the mixture was stirred at room temperature for about 3 h and then poured into ice-cold water (100 mL) with stirring. The oily layer was extracted with ethyl acetate. The dried ethyl acetate solution of the peptide was evaporated in a vacuum to get the crude peptide. The peptide was purified with 10% CH3OH/CHCl3 over a silica gel column to give 2.47 g (75%) of Boc-Ala-Asn-OCH2CH3 (2). The peptide was an amorphous solid: Rf ) 0.58 (10% CH3OH/CHCl3); IR (neat) νNH 3380 cm-1, νC)O 1725, 1672 cm-1, νC-H 2962 cm-1, νC-O (Boc) 1214 cm-1; 1H NMR (CDCl3) δ 1.39 (s, CH3Boc, 9H), 4.46 (m, R-CH-Ala), 1.44 (d, β-CH3-Ala), 4.71 (m, R-CH-Asn), 2.63, 2.76 (d, β-CH2-Asn), 7.48, 6.97 (d, δ-NH2-Asn), 4.15 (q, -COO-CH2-CH3), 1.2 (t, -COO-CH2-CH3). Peptide 3 is a kind gift from Gian Paolo Lorenzi, ETH-Zurich, Switzerland, and used as such. The fluorescence and absorbance spectra were recorded on a Perkin-Elmer LS5B spectrofluorometer and Hewlett Packard 8452A diode array spectrophotometer, respectively. In the case of peptide 1, pyrene and N-cetyl pyridinium chloride (CPC) were used as external fluorescent probe and quencher, respectively. The excitation and emission slit widths are 5.0 and 2.5 nm, respectively. In the case of peptide 2, ANS was used as the fluorescent probe. The utilization of pyrene-CPC and ANSCPC pairs for the determination of the aggregation number have been reported by a number of workers.2,7,8 The ANS was excited at 346 nm. The peak obtained at 396 nm is due to Raman scattering of water molecules.9 The concentration of pyrene and ANS was kept low (40 °C) ca. 19.7 J K-1mol-1.5 A number of observations have confirmed the view that globular proteins and their aggregates could be considered as “crystal molecules” owing to the solid-like packing of their interior.19 It has been recently shown that the convergence unfolding entropy of typical globular proteins (∆S ) 18 J K-1 mol-1) resembles the fusion entropy of small organic crystals. It is noteworthy that this value is close to the “melting” entropy calculated in the present work in the peptide aggregate (∼23.0 J K-1 mol-1). According to the present results, the structural transition of the peptide aggregate is schematized by melting of a “crystal-like” core in to a “liquid-like” state. This idea is also supported by the sudden red shift of λmax observed in the ANS spectrum (Figure 3a) and the higher temperature coefficient interior dielectric constant of the peptide aggregate (Figure 2). (19) (a) Young, L. R. D.; Fink, L. A.; Dill, K. A. Acc. Chem. Res. 1993, 26, 614. (b) Ragone, R.; Colonna, G. J. Am. Chem. Soc. 1995, 117, 16.
Structural Transition of Nonionic Peptide Aggregates
Langmuir, Vol. 15, No. 17, 1999 5471
Figure 6. Plot of difference in specific conductance (KCl probe, 1 × 10-6 M) against peptide 1 concentration at various temperatures. Figure 8. Plot of Raman scatter intensity at 396 nm against peptide 3 concentration at various temperatures. λex ) 346 nm.
Figure 9. Plot of ln(cmc/N h ) against temperature for Boc-AlaAsn-OEt aggregate.
Figure 7. Plot of Raman scatter intensity at 396 nm against peptide 2 concentration at various temperatures. λex ) 346 nm.
Aggregational Studies of Nonionic Peptides. For comparison, aggregational studies of two more nonionic peptides (2 and 3) are carried out. The plots of conductance against peptide 1 concentration at various temperatures are shown in Figure 6. It is seen that the specific conductivity of the solution was found to increase upon the onset of micellization. An explanation for the increase in conductance in the presence of structure breaking moieties has been suggested independently by Frank and Evans and by Gurney,20 and we have employed the same principle to peptide5 1. The plot of Raman scattering intensity at 396 nm against concentration of (20) Desnoyers, J. E.; Jolicoeur, C. In Modern Aspect of Electrochemistry, No. 5; Bockris, J. O. M., Conway, B. E., Eds.; Plenum Press: New York, 1969; Chapter 1, p 15.
peptide 2 has been given in Figure 7. The abrupt changes in the slope of the plots are considered as critical micellar concentration (cmc) of the peptides. The plot of the Raman scattering intensity at 396 nm against temperature for the peptide 3 aggregate is given in Figure 8. Determination of the Aggregation Number. The aggregation number N h of the peptide was calculated by measuring the quenching of a micelle bound fluorescent probe by interacting with a quencher using the expression,21,22
ln(I0/I) ) N h [Q]/(Cs - cmc)
(8)
where I0 and I are the emitted light intensities with quencher concentration being zero and [Q], respectively. N h is the mean aggregation number of the peptide, and Cs (21) Turro, M. J.; Yekta, A. J. Am. Chem. Soc. 1978, 100, 5951. (22) (a) Luo, H.; Boens, N.; Van der Auweraer, M.; De Schryver, F. C.; Mallaris, A. J. Phys. Chem. 1989, 93, 3244. (b) Mandal, A. B. J. Surf. Sci. Technol. 1985, 1, 93. (c) Hall, D. G.; Pethica, B. A. In Nonionic surfactants; Schick, M. J., Ed.; Marcel Dekker: New York, 1967; Chapter 16, pp 516-557.
5472 Langmuir, Vol. 15, No. 17, 1999
Murugesan et al.
Table 2. Critical Micellar Concentration (cmc), ∆G°m, ∆H°m, ∆S°m, and ∆C°p of Peptide 2 Aggregate in Aqueous Medium temp (K)
cmc (10-5 M dm-3)
cmc (10-6 mole fraction)
∆G°m (kJ mol-1)
∆H°m (kJ mol-1)
∆S°m (J K-1 mol-1)
294 299
11.0a 15.5a 15.1b 26.5a 36.0a 26.3a 20.1a
1.99 2.79
-35.46 -35.25
-62.52 -64.66
-92.04 -98.36
4.77 6.49 4.74 3.62
-34.47 -34.69 -36.09 -37.12
-66.85 58.31 60.20 61.34
-106.51 297.12 302.80 306.73
304 313 318 321 a,b
∆C°p (kJ K-1 mol-1) -0.433
0.392
cmc values determined by Raman scatter intensity measurements at 396 nm and conductance method.
Table 3. Critical Micellar Concentration (cmc) and Other Thermodynamic Parameters of Peptide 3 Aggregate at Various Temperatures temp (K)
cmca (10-5 M dm-3)
cmc (10-6 mole fraction)
∆G°m (kJ mol-1)
∆H°m (kJ mol-1)
∆S°m (J K-1 mol-1)
283 288 293 301 308 313
28.51 30.28 31.80 80.87 57.31 43.51
5.14 5.46 5.73 14.57 10.33 7.84
-28.65 -29.02 -29.40 -27.87 -29.40 -30.59
-7.32 -7.59 -7.85 38.79 40.62 41.95
75.37 74.41 73.55 221.46 227.34 231.76
a
∆C°p (kJ K-1 mol-1) -0.076
0.260
cmc values determined by Raman scatter method. Table 4. Calculated and Experimental ∆Str Values for the Peptide Aggregates 1, 2, and 3 nonionic peptides
∆Htr (kJ mol-1)
Tm (K)
Boc-Leu-Asn-OEt (1) Boc-Ala-Asn-OEt (2) Boc-(Ile)2-NH(CH2CH2O)3-CH3 (3)
7.2 126.08 46.64
313 310 297.5
∆Str (J K-1 mol-1) calcd exptl 23 403.74 156.77
27.73 403.63 147.91
Figure 11. Plot of ANS fluorescence intensity against temperature for peptide 2 aggregate: [peptide 2] ) 1 × 10-3 M (fixed); [ANS] ) 1 × 10-6 M (fixed); λex ) 345 nm, λem ) 426 nm. Figure 10. Plot of ln(cmc) against temperature for Boc-(Ile)2NH(CH2CH2O)3CH3 aggregate.
is the total concentration of the peptide. From the slope of the plot of ln(I0/I) against the quencher concentration, the aggregation number of peptide 2 is found to be 4 and that of peptide 1 is reported to be 19.5 The aggregation number for peptide 3 could not be obtained by the fluorescence quenching method. The various thermodynamic parameters for the peptide aggregates are calculated by determining their cmc values at various temperatures (Figures 9 and 10).22c The critical micellar concentration (cmc), ∆G°m, ∆H°m, and ∆C°p of peptides 2 and 3 are given in Tables 2 and 3, respectively. The structural transition temperatures of the peptide 2 and 3 aggregates were determined by ANS fluorescence (Figure 11) and Raman scattering intensity measurements (Figure 12), respectively.
The different Tm values for peptides 1, 2, and 3 aggregates are given in Table 4. The changes in the enthalpy and entropy upon structural transition are also furnished in Table 4. The cmc of peptide 2 increases with the increase in temperature from 294 to 304 K indicating that the aggregation is hindered on increasing the temperature. Also, the negative value of ∆H°m for peptide 2 indicates a hydrogen-bonded aggregate at low temperatures. However, on increase of the temperature above 313 K, the cmc decreases, indicating that the aggregate is entropically stabilized. Therefore, it is inferred that in this temperature range, the aggregate “melts” resulting in the formation of more “free” peptide monomers in the aggregate. Comparative Studies on the Aggregate Forming Characteristics and Structural Transitions of the Peptides 1, 2, and 3. A linear relationship exists between the increase in entropy and a compensating increase in
Structural Transition of Nonionic Peptide Aggregates
Figure 12. Plot of Raman scatter intensity at 396 nm against temperature for peptide 3 aggregate; λex ) 345 nm; [peptide 3] ) 1 × 10-3 M (fixed).
enthalpy of the transition. As ∆Htr increases, the ∆Str also increases, ensuring the transition temperature is neither larger nor smaller. This kind of compensation has long been recognized,23 and it was interpreted that this behavior is a consequence of the properties of liquid water. If the transition is assisted by water, then the “melting” will result in more “free” water molecules resulting in a larger value of ∆Str as in the case of peptide 1 and 2. The (23) (a) Lumry, R.; Rajender, S. Biopolymers 1970, 9, 1125-1127. (b) Lemieux, R. U.; Delbaeret, L. T. J.; Beierbeck, H.; Spohr, U. In HostGuest Molecular Interactions: from Chemistry to Biology; Wiley: Chichester (Ciba Foundations Symposium 158), 1991; pp 231-248. (c) Effink, M. R.; Anusiem, A. C.; Biltonen, R. L. Biochemistry 1983, 22, 3884-3896.
Langmuir, Vol. 15, No. 17, 1999 5473
enthalpy compensation results from the disruption of hydrogen bonding in water due to the transition. Hence it appears when compared to peptide 1, in peptides 2 and 3 solvent seems to play a dominant role in the aggregation and structural transition. Peptide 1 seems to form better aggregates in terms of higher aggregation number. This is attributable to the presence of isobutyl moiety, which is capable of forming more flexible aggregates. More flexibility with higher hydrophobicity leads to stronger aggregation.19 In the case of peptide 2, the cmc is comparable to peptide 1, with a very small aggregation number (N h ), and this has been attributed to the smaller size of the side chain. In peptide 3, despite the two hydrophobic isoleucine moieties, the presence of three oxyethylene groups which are capable of acting as hydrophilic groups, better packing and greater flexibility in the aggregate forming characteristics are observed. The nature of this aggregate seems to be different from that obtained from peptides 1 and 2. This could be observed from the pyrene-binding studies of peptide 3. Pyrene did not bind to the peptide 3 aggregate, indicating a “tight” core with flexible hydrophilic headgroups. These studies indicate that depending upon the nature of the sequence and polarity, the aggregate forming characteristics are altered. Acknowledgment. We are grateful to Dr. T. Ramasami, Director, CLRI, for his constant encouragement. We are also thankful to Dr. V. Durai and Dr. P. Ramamurthy for stimulating discussion and their kind help to carry out most of the experiments presented in this work. The author M.M. thanks CSIR for financial assistance in the form of Junior/Senior research fellowships. LA9814079
