Leucylglycine Oligopeptides and Their Aggregational Behavior in

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J. Phys. Chem. B 2008, 112, 5824-5833

Folded Structures of L-Leucylglycine Oligopeptides and Their Aggregational Behavior in Aqueous Solution: Raman Scattering Spectra and Proton NMR Spin-Lattice Relaxation Studies Aki-Hiro Yoshino,† Hiro-Fumi Okabayashi,*,† Hide-Hiro Kanbe,† Keita Suzuki,† and Charmian J. O’Connor‡ Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi 466-8555, Japan, and Department of Chemistry, The UniVersity of Auckland, PriVate Bag 92019, Auckland, New Zealand ReceiVed: June 18, 2007; In Final Form: February 9, 2008

The aggregational behavior of three L-leucylglycine oligopeptides (residue numbers of glycine are 3, 4, and 5) in aqueous solution was investigated by the use of Raman scattering and 1H NMR spin-lattice relaxation methods. The results indicate that their oligopeptides take up a folded structure to form dimeric aggregates above their critical aggregation concentration. The application of one-dimensional aggregate theory to these systems provides the following prediction. Elongation up to 6 glycine residues makes it possible to form dimeric aggregates, but further elongation (up to 7 glycine residues) makes the aggregates very unstable, and up to 8 or 9 glycine residues makes the formation of dimeric aggregates very difficult. The one-dimensional aggregate theory may be used to predict the existence of peptide aggregates through intermolecular hydrogen bonding.

Introduction Understanding the nature of the steric structural change of molecules upon their aggregation or dissociation contributes fundamental knowledge to an investigation of the relationship between the functional appearance and the conformational change of biological molecules. In fact, conformational studies of simple molecules, which form aggregates in solution, support our understanding of the physicochemical properties of biological systems with respect to their relationship to an aggregated structure. We demonstrated that one specific isomer, of all the possible rotational isomers about the CH2-CH2 (or CH2-CH) single bond1-5 or the peptide C-N bond6 with double bond character (30-40%), is preferentially stabilized upon aggregation in water. These results provided clear evidence that the hydrophobic interaction is the only driving force for such a structural change. Furthermore, it was confirmed that N-acylglycine oligomers7,8 form their aggregates to stabilize a specific isomer in whose formation hydrogen bonding, in addition to a hydrophobic interaction, plays a critical role in its stabilization. Becker et al.9 and James et al.10 indicated that the selfassociation of N-urethanyl amino acids may be important in enzymatic and synthetic reactions. Recently, Okabayashi et al.11 reported IR and Raman scattering studies of N-acetyl-L-glutamic acid oligomeric benzyl esters (residue number Np ) 4, 5, 6, 8, 10, 12, and 14). The results showed that a transition from the random form to an antiparallel-type β-sheet structure occurs above the critical aggregation concentration (cac) and that the population of this sheet structure increases with an increase in concentration. Moreover, Ishida et al.12 elucidated the aggregational behavior of these oligopeptides by use of small-angle * Corresponding author. E-mail: [email protected]. † Nagoya Institute of Technology. ‡ The University of Auckland.

neutron scattering (SANS) and small-angle X-ray scattering (SAXS) spectra. Consequently, it has been pointed out that the driving force for starting the aggregational pattern may originate from the side chain-side chain interactions. Proteins are compacted into giant self-assembled aggregates. Considerable attention has been given to structural studies of histones and various histone complexes as giant aggregates.13 Indeed, it has been found that a conformational change upon the formation of a giant aggregate occurs in complexes of histones and that conformational changes in the core histone complex are reversible.14 These studies serve as a pathway not only toward the complete understanding of chromatin structure but also toward an understanding of structural changes occurring in vivo. It is well-known that the folded conformational portions contribute strongly to self-assembled structures of proteins.15 In such a folded conformational portion, a region termed a β-turn16 exists in which the polypeptide chain reverses its direction by ca. 180°. This region contains three peptide groups at the turn. Venkatachalam17 proposed three main types of β-turns (types I, II, and III) and their mirror images for such structures. Further analysis18 has indicated the existence of five additional types of turn structures in proteins. The three main types (I, II, and III) have, in common, an intramolecular hydrogen bond between the peptide CO group of the first residue (residue 1) and the NH group of the fourth residue (residue 4).17 Types I and II take up a non-helical structure, while type III corresponds to one turn of the 310-helical structure. These β-turn structures furnish a suitable model for investigating the short-range interactions between the side chain and the backbone, which play an important role in the aggregation process of proteins.19-21 A number of conformational studies of short peptides, as models of a β-turn, have so far been carried

10.1021/jp074732q CCC: $40.75 © 2008 American Chemical Society Published on Web 04/12/2008

Folded Structures of L-Leucylglycine Oligopeptides

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SCHEME 1

Figure 1. Observed recovery data (10 points) of magnetization (Mz, dimensionless) and calculated curves for the Gly5 resonance peak of LG5 (2, C ) 0.029 mol/L and b, C ) 0.066 mol/L).

out.22-24 However, further studies of the property of this β-turn structure are highly desirable, to elucidate the role of the shortrange interactions in proteins. The β-turn structures of model compounds already have been identified and solved by X-ray diffraction analysis.25-27 The structure of a single crystal of L-leucyl-triglycine has been accurately solved by X-ray structural analysis,28 and the similarity of its folded structure to a type II β-turn already has been identified. L-Leucylglycine oligopeptides (numbers of glycine residues are 3, 4, and 5), which contain the tertiary butyl chain as a hydrophobic group and the peptide amino or carboxyl groups as hydrophilic groups, may be regarded as one species of a surfactant molecule. In this present study, the aggregational behavior of surface-active oligopeptides in aqueous solution has been studied by use of Raman scattering and proton NMR spinlattice relaxation methods. In particular, a one-dimensional aggregate model29 is applied to these aggregate systems, and the relationship between the number of peptide residues and the bond energy (that is, intermolecular hydrogen-bonding energy) required to form the aggregates is discussed. Experimental Procedures Materials. L-Leucyl-triglycine (LG3), L-leucyl-tetraglycine (LG4), and L-leucyl-pentaglycine (LG5) (numbers of peptide residues Np are 3, 4, and 5) (Scheme 1) were synthesized by a stepwise procedure, as follows. Benzyl esters of N-t-butoxycarbonylglycine (N-t-BOC-glycine) trimers, tetramers, and pentamers were prepared from N-t-BOC-glycine trimers, tetramers, and pentamers. The usual 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) method30 was used for these condensation reactions in the presence of equimolar 1-hydroxybenzotriazole. The BOC groups of these oligomeric benzyl esters were removed by the action of trifluoroacetic acid. Benzyl esters of these BOC-free oligomers were coupled with BOC-L-leucine (BOC-L-Leu). The BOC and benzyl groups of BOC-L-leucylglycine oligomeric benzyl esters (BOC-L-Leu-(Gly)N-O-benzyl, N ) 3, 4, and 5) were removed in anhydrous HF. These crystalline oligopeptides were prepared by the same procedure used in preparation of the crystals needed for the X-ray diffraction analysis of LG3.28

Raman Scattering Measurements. All Raman scattering spectra were obtained with a Nicolet 950 Fourier transform Raman spectrometer (4000-100 cm-1) using a Nd:YAG laser (CVI) excitation wavelength of 1064 nm with a resolution of 4 cm-1 at 25 °C under laser power of 800-1200 mW. The reproducibility of the Raman frequencies was 0.01 cm-1. The reported frequencies were accurate to (1 cm-1 for sharp bands and (2-3 cm-1 for broad and weak bands. The Raman scattering spectrum of the solvent (H2O) was successfully subtracted from all Raman spectra of the sample solutions in the region of 1500-1800 cm-1 since Raman bands of water in this region are relatively weak. The program attached to the Raman spectrometer was used to obtain the heights of the Raman scattering bands at 1030 and 1650 cm-1 for calculation of the relative peak heights (I1650/I1030). The sample solutions for measurement of the Raman spectra of these oligopeptides were prepared by dissolving a weighed sample of an oligopeptide in 0.1 mL of H2O at 25 °C. The clear solutions were sealed in capillary cells. 1H NMR and Spin-Lattice Relaxation Time Measurements. 1H NMR spectra were recorded on a Varian XL-200 spectrometer operating at 199.975 MHz (spectral width of 3000.3 Hz, 32 768 points in the time domain, acquisition time of 2.56 s, and delay of 4.00 s). The 1H chemical shifts (δ, ppm) are given relative to the external standard (capillary of dioxane(CH3)3Si(CH2)3SO3Na (DSS)-D2O solution) (the 1H resonance peak of DSS at the highest field was regarded as a reference). The oligomer-D2O (CEA, isotopic purity of 99.8%) solutions with different concentrations (2-148 mM/L) were placed into NMR sample tubes, which were sealed, and the contents were homogenized by shaking. The 1H spin-lattice relaxation times (T1, s) were measured by the inversion-recovery Fourier transform method (π-τ-π/2 sequence) with a spectral width of 1377.8 Hz, acquisition of 11.892 s, delay of 2.110 s, π/2 pulse-width of 12.5 µs, and 4 times spectrum accumulation. The pulse repetition time (tpr) was chosen so as to satisfy the relation tpr g 5T1. The monoexponential recovery data of 10 points were observed for each resonance signal using the program attached to the NMR spectrometer. The curve best-fitted to the observed data was calculated by varying the three parameters (T1, Mz (magnetization) at t ) 0 s and Mz at t ) ∞ s). The observed data of 10 points and calculated recovery curves for the Gly5 resonance peak of LG5 as representatives are shown in Figure 1. The T1 value used to calculate the best-fitted curve was regarded as the observed T1 value.

5826 J. Phys. Chem. B, Vol. 112, No. 18, 2008 Abbreviations for Assignment of Vibrational Spectra. The following abbreviations are used for the assignment of the vibrational spectra: amide I, mainly CdO stretching mode; amide II, NH in-plane bending mode coupled with amide CN stretching mode; amide III, amide CN stretching mode coupled with N-H in-plane bending mode and CR-H bending modes;31 asym. bend, asymmetric bending mode; wag, wagging mode; skel. str., skeletal stretching mode; and rock, rocking mode. Numbering of Glycine Residue and Peptide Group. The numbering of glycine residues and peptide groups for these oligopeptides is shown in Scheme 1: for LG3, Leu-Gly1-Gly2Gly3; for LG4, Leu-Gly1-Gly2-Gly3-Gly4; and for LG5, LeuGly1-Gly2-Gly3-Gly4-Gly5.

Yoshino et al. TABLE 1: Raman Bands (cm-1) Characteristic of the LG3-Type Folded Structure Observed in Common for Aqueous LG3, LG4, and LG5 Solutionsa and Raman Bands (cm-1) of Crystalline LG3 Taking a Folded Structure aq LG3, LG4, and LG5 Raman shifts (cm-1)

crystalline LG3 Raman shifts (cm-1)

assignment

1683-1684 1615-1617 1530-1540 1472-1473 1449 1418-1419 1400-1402 1346 1302-1309 1267-1272 1133-1134 1029-1030 961-962 917 887 848-852

1679 1613 1545 1463 1449 1412 (1402) (IR) 1347 1300 1265 1132 1028 961 916 888 849

amide I amide I amide II leucine CH3 asym. bend CH2 bend CH2 bend + wag CH2 wag amide III amide III amide III skel. str. skel. str. CH2 rock CH2 rock CR-C and C-N stretch CR-C and C-N stretch

Results and Discussion In the previous paper,32 the Raman scattering and infrared (IR) absorption spectra were used to investigate the molecular conformations of the L-leucylglycine oligopeptides (Np ) 3, 4, and 5) in the crystalline state and aqueous solution. Results of special relevance to this present study may be summarized as follows. The Raman scattering and IR absorption spectra of LG4 and LG5 in the crystalline states were compared to those of LG3, in which a folded structure already was confirmed by X-ray diffraction analysis.28 Consequently, in their solid states, the LG4 oligopeptide takes up a folded structure similar to that of LG3, while the LG5 oligopeptide adopts a helical structure similar to that of polyglycine II.33 Therefore, the leucyl residue exerts a folded structure effect on the tetraglycine portion of LG4 as well as on the triglycine portion. The Raman scattering spectra of the two aqueous (dilute and concentrated) solutions for these oligopeptides provide evidence that the folded structure of LG5, as well as that of LG3 and LG4, is stabilized in a concentrated solution. The purpose of the present study was to examine the properties of the folded structure of the oligopeptides in aqueous solution and to predict the residue number of further longer L-leucylglycine oligopeptides, making folded structures possible in aqueous solution. Raman Scattering Spectra of LG3, LG4, and LG5 and cac. We investigated in further detail the concentration dependence of the Raman spectra for the aqueous solutions of the three oligopeptides. For the Raman spectra of these oligopeptides in concentrated aqueous solution, the observed Raman shifts (cm-1) only of medium and strong bands, characteristic of an LG3-type folded structure,32 are listed in Table 1, together with their assignment. In particular, the focus in this present study is on the amide I mode (1600-1700 cm-1) that is sensitive to the conformation and the skeletal stretching mode (1029-1033 cm-1). In the amide I region, for the concentrated aqueous samples (1.17-1.28 mol/L), some very weak bands, in addition to the strong bands at 1683-1684 cm-1 arising from the folded structure, were found at 1615-1617, 1629-1640, and 16701673 cm-1 (not listed in Table 1) and are assigned to other conformations with small populations. These observations indicate that, although the folded structure similar to that of the crystalline LG328 is predominant, other conformations with small populations also coexist. For the dilute aqueous samples (0.12-0.17 mol/L), however, very broad amide I bands were observed at 1644 cm-1 for LG3 and LG4 and at 1648 cm-1 for LG5 (not shown here), which closely correspond to the amide I mode (1646 cm-1) of a type III β-turn calculated by Krimm

a

C: 1.28, 1.17, and 1.21 mol/L, respectively.

et al.33,34 This observation may imply the existence of the type III conformation in dilute solution. Figure 2 shows the Raman scattering spectra of LG3, LG4, and LG5 in aqueous solution in the region of 1500-1800 cm-1, with emphasis being placed on the very dilute concentration region. For LG3 (Figure 2a), we find that the intensity of the amide I band at 1684 cm-1 decreases with the reduction of concentration until it finally disappears at lower concentrations. This observation indicates that the populations of the folded structure, stabilized preferentially in the more concentrated solutions, become smaller with a decrease in concentration. For LG4 and LG5, similar observations also were made, and representative spectra of the dilute and concentrated sample solutions are shown in Figure 2b. The very broad amide I bands are observed, in common, at ca. 1650 cm-1 for very dilute solutions of the three oligopeptides. These bands at 1650 cm-1 may be assigned to superimposed amide I modes of various conformations (that is, random forms) coexisting in the dilute solutions, in which the structures of the solutes may be regarded as disordered states. The previous information suggests that aggregation of these oligopeptides in concentrated solution results in the preferential stabilization of the folded structure, constituting a more ordered state, leading to an increased intensity of the 1684 cm-1 band and, as a consequence, a decreased intensity of the 1650 cm-1 band. To determine the critical concentration for such a disorderorder transition, we used the ratio (I1650/I1030) of the peak height of the 1650 cm-1 band relative to that of the 1030 cm-1 band. For LG3, we examined the concentration dependence of the height of the 1030 cm-1 band. It was found that the height of the Raman band at 1030 cm-1 as a function of the concentration provided a good linear relationship. Therefore, the use of the 1030 cm-1 band as a reference is well-suited to evaluate the concentration dependence of the relative Raman scattering intensity of the amide I mode. Figure 3 shows plots of the relative intensity (IR ) I1650/I1030) versus concentration (C, mol/L) for LG3, LG4, and LG5. For these oligopeptides, the relative intensity tends to decrease with an increase in concentration. However, it is found that points of inflection appear in the plots: the concentrations correspond-

Folded Structures of L-Leucylglycine Oligopeptides

J. Phys. Chem. B, Vol. 112, No. 18, 2008 5827

Figure 2. (a) Concentration dependence of the Raman scattering spectrum of LG3 in the 1500-1800 cm-1 region and (b) Raman scattering spectra of dilute and concentrated LG3, LG4, and LG5 solutions in the 1500-1800 cm-1 region.

Figure 3. Concentration dependence of the relative peak height IR (I1650/I1030) for LG3, LG4, and LG5. The straight lines below and above the concentration corresponding to the inflection point were obtained by linear least-squares fits.

ing to the inflection points are 0.126 mol/L for LG3, 0.079 mol/L for LG4, and 0.048 mol/L for LG5. The appearance of such inflection points reflects the transition from a disordered state to an ordered state. The concentrations corresponding to the inflection points may be regarded as the cac, at which the beginning of the conformational transition from the random forms to the LG3-type folded structure occurs. Indeed, for sodium N-acylsarcosinates6 and for N-acylglycine oligomers,8 it has been confirmed that the conformational change

about the peptide C-N bond occurs at the critical micelle concentration (cmc). Furthermore, we should note that the cac value decreases linearly with an increase in glycine residue number (N ) -25.3(cac) +7.0). This tendency is very similar to the case of surfactants in aqueous solution,35 in which the cmc becomes smaller with an increasing number of CH2 groups. 1H NMR Spectra of LG , LG , and LG in D O and 1H 3 4 5 2 Spin-Lattice Relaxation Rates. For the 1H NMR spectra of LG3, LG4, and LG5 in D2O solution, the resonance peaks of the protons belonging to the N-terminal L-leucyl residue were observed in common. Assignment of these peaks was made by reference to assignment of the proton NMR spectra (60 MHz) of LG3 and LG4 in D2O solution, already reported by Beecham and Ham.36 In the 1H NMR spectrum (Figure 4) of LG3 (0.06 mol/L) in D2O solution, the 1H resonance peaks at 0.95 ppm were assigned to the L-leucyl (CH3)2 protons. The peaks at 1.78 and 1.70 ppm arose from the leucyl βCH2 and γCH protons, respectively. For LG4, the resonance peaks at 0.95, 1.78, and 1.71 ppm were assigned to the L-leucyl (CH3)2, βCH2, and γCH protons, respectively. For LG5, the resonance peaks at 0.95, 1.78, and 1.71 ppm were assigned to the (CH3)2, βCH2, and γCH protons, respectively, from analogy with assignment of these protons in LG3 and LG4.36 The expanded resonance spectra of the L-leucyl RCH and glycyl CH2 protons in the 3.6-4.2 ppm region are shown in Figure 5. The resonance peaks at 4.15, 3.92, and 3.77 ppm for LG3 were assigned to the CH2 protons of Gly1, Gly2, and Gly3,

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Figure 4. 1H NMR spectrum of the LG3-D2O solution (0.135 mol/L).

Figure 5. Expanded 1H NMR spectra of the LG3 (0.095 mol/L)-, LG4 (0.078 mol/L)-, and LG5 (0.048 mol/L)-D2O solutions in the region of 3.6-4.2 ppm.

respectively, on the basis of the assignment of LG3 already made.36 In the spectrum of LG4, the resonance peaks at 3.77 and 3.96 ppm were assigned to the CH2 protons of Gly4 and Gly3, respectively, and the newly appearing peak (marked with an asterisk) at 3.97 ppm was assigned to Gly2. For LG5, the peaks at 3.77 and 3.93 ppm were assigned to Gly4 and Gly5, respectively, and the peak at 3.97 ppm, which closely corresponds to the peak of Gly2 for LG4, was assigned to the CH2 protons of Gly2 of LG5. Accordingly, the newly appearing resonance peak (marked with a double asterisk) at 3.95 ppm must be assigned to the CH2 protons of the Gly3 residue of the LG5 molecule. We may expect that aggregation reduces the mobility of an oligopeptide molecule as a whole and its segmental mobility. The spin-lattice relaxation time (T1, s) of interacting dipoles

is affected by their relative motion, and therefore, T1 of dipoles on a molecular segment becomes an indicator of segmental mobility.37 If protons associated with a particular resonance of monomers exchange magnetization rapidly on the T1 time scale with protons associated with the same resonance of aggregates, then the observed relaxation rate R1 (s-1) may be regarded as a weighted average of the R1 value (R1m) in the monomeric state and that (R1A) in the aggregated state. When oligopeptide molecules are in the monomolecular state, the contribution of R1m becomes predominant since the effect of interoligopeptide interactions is expected to increase the correlation time (τc). Aggregation of the molecules makes the contribution of R1A greater. When the conformational change of an oligopeptide molecule occurs upon aggregation, the contribution of R1A becomes greater. In the present study, the concentration dependence of the R1 value for each segment of the oligomeric molecule was measured, and the difference in the segmental mobility among the different residues was examined. Figure 6a shows the concentration dependence of R1 (s-1) for each 1H resonance peak of LG3. For the resonance peaks of all segments, it was found that the values of R1 increase with an increase in concentration. In particular, a rapid increase in the R1 value occurs above the concentration 0.122 mol/L, which corresponds closely to the cac of LG3 obtained from the Raman spectra (termed the amide I method). It is reasonable to assume that the T1 values involved are on the high-temperature side of their T1 minima, so that such an increase in R1 implies that aggregation induces the reduction of the mobility of a LG3 molecule as a whole. We note that the extent of an increased R1 value depends strongly upon the position of the residue: the magnitudes of the R1 value increase at the cac are 0.08 s-1 for Gly1, 0.06 s-1 for Leu-RCH, 0.03 s-1 for Gly2 and Leu Me (C(CH3)2), and 0.04 s-1 for Gly3. Therefore, of the four segments of LG3, the change in mobility of Gly1 is the largest, followed by that of Leu-RCH. This behavior will be due to the stabilization of the folded structure upon the formation of aggregates. Furthermore, when we compare the slopes of the two straight lines above and below 0.122 mol/L, it is found for all resonance peaks of LG3 that the slopes in the concentrated region are larger than those in the dilute region. This fact implies that the effective correlation times of the LG3 molecule are increasing more

Folded Structures of L-Leucylglycine Oligopeptides

Figure 6. Concentration dependence of R1 (s-1) for each 1H resonance peak of LG3 (a), LG4 (b), and LG5 (c). The straight lines below and above the concentration corresponding to the inflection point were obtained by linear least-squares fits.

rapidly with an increasing concentration in the region above the cac than in the region below the cac. Figure 6b shows the concentration dependence of the R1 value for LG4. For the resonance peaks of all residues, a rapid increase of the R1 value occurs above a concentration of 0.072 mol/L, which corresponds closely to the cac of LG4 determined by the amide I method. For LG4, the magnitudes of the increase in the R1 value at the cac are 0.1 s-1 for the Leu-CH, Gly1-, and Gly2-CH2 groups

J. Phys. Chem. B, Vol. 112, No. 18, 2008 5829 and 0.07 and 0.04 s-1, respectively, for the Gly3- and Gly4CH2 groups. Therefore, the change in mobility of the Leu-RCHGly1-Gly2 segment is the largest, followed by reduction in the mobility of the Gly3 and Gly4 residues. We may assume that preferential stabilization of the folded structure of LG4, upon aggregation, brings about the low flexibility of the Leu-RCHGly1-Gly2 segments. When we compare the slopes of the straight lines above the cac, we see that the slope for the Gly1 residue is the largest. This result may be due to preferential stabilization of the folded structure upon aggregation, probably indicating that the formation of an aggregate affects strongly the mobility of the Gly1 residue. The increase in R1 values at the cac and the slopes of the straight lines for all the peaks of LG4 are clearly larger than those for LG3. Since τc for tumbling motion is expected to increase as the size of the molecule increases, and T1 is on the high-temperature side of its minimum (i.e., R1 increases as τc increases), the former can be understood, at least in part, in terms of a larger change in τc expected as the larger LG4 units aggregate. The latter may imply that some regions of the LG4 aggregates are more tightly formed than the corresponding regions of the LG3 aggregates. Figure 6c shows the concentration dependence of the R1 value for LG5. In the R1 versus C plots, the change in the R1 value occurs for the Gly1-CH2 and Leu-RCH protons at a concentration of ca. 0.043 mol/L, which corresponds closely to the cac determined by the amide I method: the extent is 0.01 s-1 for Gly1-CH2 and less than 0.01 s-1 for Leu-RCH. This observation indicates that the Leu-RCH-Gly1 segment also is restricted upon aggregation, caused by preferential stabilization of the folded structure. The weak inflection at cac for Gly2 may be caused by stabilization of the folded structure, which causes the Gly2 residue to become rigid. For the residues of Gly4 and Gly5, very weak inflections are seen in the plots of R1 versus C, implying that these segments are also restricted upon aggregation, but only to a small extent. For the segments of Gly3, the inflection point at the cac almost disappears in the R1 versus C plots, indicating that the mobility of this segment is almost unrestricted, even above the cac. For the C(CH3)2 protons, there is no inflection point in the R1 versus C plots, indicating that formation of the LG5 aggregates does not affect the mobility of the tertiary butyl chain. This result may indicate the absence of hydrophobic interactions between the two hydrocarbon chains in the aggregate. Thus, it is found that restriction of the Leu-RCH and Gly1 segments occurs, in common, for LG3, LG4, and LG5 above the cacs. Accordingly, we may assume that stabilization of the LG3-type folded structure upon aggregation induces a strong restriction of these segments and that these segments play, in common, a critical role in the formation of the aggregates of these oligopeptides. The jump in R1 at the cac implies that a significant fraction of the total number of monomers at the cac forms aggregates (i.e., the concentration of monomeric molecules just above the cac is already much less than the cac since a sufficient number of monomers must already have aggregated in order for any sudden increase in R1 to be observable). The R1 contribution from the tumbling motion would increase with an increase in τc, which would increase with an increase in the size of the molecule or aggregate unit. Thus, the jump in R1, as the aggregate forms, would be larger for LG5 than for LG4 and larger for LG4 than for LG3, assuming that everything else remains constant. We see that these are competing effects

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as far as the R1 increase at the cac is concerned. So perhaps, as the size of the oligopeptide increases from LG3 to LG4, a point close to the maximum in the R1 increase at the cac is reached, and for LG5, the number of aggregates formed at a relatively low C value close to the cac may be too low to generate a step in the R1 versus C plots. We may expect that intramolecular hydrogen bonds, in addition to intermolecular hydrogen bonding, contribute strongly to the stabilization of the folded structure and the formation of aggregates. However, it is difficult to discuss the independent contributions of the intra- and intermolecular hydrogen bonds to the R1 values. Determination of Aggregation Numbers. The 1H spinlattice relaxation rates (R1, s-1) of the Gly1-CH2 protons for LG3, LG4, and LG5, measured above the cacs, were used to determine the aggregation numbers of their aggregates, based on the assumption that the aggregation reflects especially the relaxation behavior of this Gly1 residue. In the aqueous solutions of these oligopeptides above the cacs, we assume a single aggregate in equilibrium with monomers and apply the mass action law

nS S Sn

(1)

[Sn]/[S]n ) K

(2)

where S is a monomer ([S] is the concentration of monomers), Sn is an aggregate ([Sn] is the concentration of aggregates), n is the aggregation number, and K is the equilibrium constant. In the mass action law model, as distinct from the phase separation model, we do not have to regard the value of [S] as a constant above the cac. Indeed, the jumps in values of R1, observed at the cac for proton groups at various resonance positions (Figure 6), strongly suggest that the larger number of aggregates formed at the cac produce an observable increase in R1 through the effect of R1A. It is still true, however, that eq 3 remains valid

n[Sn] ) C - [S]

(3)

where C is the initial concentration of the solute. The combination of eqs 2 and 3 provides the following equation:

log(C - [S]) ) n log[S] + log(nK)

(4)

Accordingly, the slope in the plots of log [S] against log(C [S]) is equal to the aggregation number n. Since the 1H spin-lattice relaxation rates (R1) observed above the cac are equal to the weighted averages of the spin-lattice relaxation rates (R1m) of monomers and those (R1A) of aggregates, R1 can be expressed by the following equation:

R1 ) ([S]/C)R1m + [(C - [S])/C]R1A

(5)

The values of R1 for Gly1-CH2 protons, observed below the cac, increase linearly with an increase in concentration (Figure 6), reflecting the intermolecular interactions in the monomeric state. These linear relationships may be expressed by the equation R1 ) rC + q (where the constant (slope) r is the variation of R1 per unit concentration, C is the concentration of the solute below the cac, and q is equal to R1 at C ) 0), obtained by the linear least-squares fits of the R1 versus C plot below the cac. For the oligomeric solutions above the cac, we may assume that the observed R1 values contain the contribution of intermolecular interactions, which increase with an increase in C,

and that (R1′) of aggregates. On the basis of the assumption that the contribution of the intermolecular interactions follows the equation R1 ) r(C - cac) (where C g cac), the R1′ value can be expressed by the equation R1′ ) R1 - r(C - cac). The values of R1m can be obtained from the R1 value at each cac for LG3, LG4, and LG5 (Figure 6). The value of R1A can be obtained from the extrapolation of R1′ to 1/C in the R1′ versus 1/C plot (not shown). The values of R1m and R1A for LG3 are 1.741 and 2.141 s-1, respectively, 1.584 and 2.054 s-1, respectively, for LG4, and 1.756 and 2.046 s-1, respectively, for LG5. The concentration of monomers [S], needed in eq 4, can be calculated using the equation

[S] ) [(R1A - R1′)/(R1A - R1m)]C

(6)

The plots of log[S] against log(C - [S]) for LG3, LG4, and LG5 are shown in Figure 7. An aggregation number of n ) 2.1 for LG3 was obtained from the slope of this plot, and then the equilibrium constant K ) 13.2 was calculated from the intercept. Similarly, values of n ) 2.2 and K ) 18.7 for LG4 and n ) 2.0 and K ) 21.8 for LG5 were obtained. Thus, we may assume that the oligopeptide molecules of LG3, LG4, and LG5 form dimeric complexes in the relatively dilute concentration range above the cacs. Most likely, the formation of these dimeric aggregates occurs competitively at the cac, resulting in a jump of the R1 value. However, in more concentrated solutions, the existence of aggregates with aggregation numbers larger than 2 is probably possible. In fact, in the Raman scattering spectra of the concentrated solutions (C ) ca. 1.2 mol/L) of these oligopeptides,8 the scattering intensity of the amide I band at 1684 cm-1 arising from the folded structure is markedly intensified, and that at 1650 cm-1 (due to the random forms) is observed as a weak shoulder. Models of Dimeric Aggregates. Figure 8a shows a schematic model38 of the folded LG3 molecule in which the two peptide groups completely fold back on themselves at the middle Gly1Gly2 link. We may assume that the folded structural portion is extremely restricted and that the two peptide groups (peptide groups 1 and 2) are in the positions in which they easily form intermolecular hydrogen bonds with those of the other oligopeptide molecules. The following assumptions were made for the dimeric aggregates: (1) The folded conformations are stabilized, in common, in the LG3, LG4, and LG5 molecules upon aggregation (based on Raman scattering data). (2) The oligomeric molecules taking up a folded structure form dimeric aggregates above the cac, which are linked at peptide group 1 sandwiched by the leucyl-RCH and Gly1-CH2 segments through intermolecular hydrogen bonds (based on the R1 data). Figure 8b, part i shows the dimeric aggregate model of LG3, in which the two molecules taking up the folded structure are parallel to each other. In this model, the peptide group 1, sandwiched by the Gly1-CH2 and Leu-RCH segments, participates in the formation of the dimeric aggregates through intermolecular hydrogen bonding (shown by bi-arrows). Furthermore, hydrophobic interactions between the two tertiary butyl chains, due to the aggregation, are possible in this parallel model. The C-terminal Gly3 residue may participate in the formation of intramolecular hydrogen bonds to stabilize the folded structure, resulting in the restricted states of the Gly2and Gly3-CH2 segments. We may assume the existence of intramolecular hydrogen bonds in aqueous solution, as shown by the broken lines. Indeed, X-ray structural analysis has confirmed the existence of strong intramolecular hydrogen

Folded Structures of L-Leucylglycine Oligopeptides

J. Phys. Chem. B, Vol. 112, No. 18, 2008 5831

Figure 8. Schematic model of the folded LG3 molecule38 (a) and the aggregate models (b) of LG3 (i), LG4 (ii), and LG5 (iii) (the trans form for a peptide group CONH was assumed). The bi-arrows denote the intermolecular hydrogen bonds assumed between each peptide group 1 in the oligopeptide. Broken lines imply the assumed presence of intramolecular hydrogen bonds.

Figure 7. Plots of log[S] against log(C - [S]) for LG3 (a), LG4 (b), and LG5 (c).

bonding between the amide NH and the terminal carbonyl oxygen atom in crystalline triglycine.39 The antiparallel model cannot explain the experimental observation that the leucyl (CH3)2 protons are restricted upon aggregation, although such may be possible on a molecular structural level. However, the existence of an equilibrium between the parallel type and an adjacent antiparallel type may be possible. For LG4, we present a parallel-type model similar to that of LG3, as shown in Figure 8b, part ii. The peptide group 1 may

participate in formation of intermolecular hydrogen bonds to tighten the dimeric aggregate. Formation of additional intramolecular hydrogen bonds, followed by the addition of Gly4, may stabilize the folded structure, bringing about the very low flexibility of the LG4 dimeric aggregate. In the antiparallel model for LG5 (Figure 8b, part iii), the Leu-RCH and Gly1-CH2 segments become restricted upon preferential stabilization of the folded structure to form the dimeric aggregates, bringing about reduction of the mobility of these segments. The peptide group 1 participates in the formation of the dimeric aggregates through a weak intermolecular hydrogen bond. However, the intramolecular hydrogen bonds that restrict the mobility of the Gly3 segment may be absent or very weak, making this segment very mobile. Weak intramolecular hydrogen bonds between peptide groups 4 or 5 and C-terminal COOH groups may be possible in this antiparallel model. Prediction of Intermolecular Hydrogen-Bonding Energy To Form the Dimeric Aggregates. In our previous paper,12 SANS and SAXS studies of the ANpZ-dioxane system provided evidence that there exists a linear relationship between the intermolecular hydrogen-bond energy per peptide residue and the residue number of peptides. From this linear relationship, it was found that the energy of intermolecular hydrogen bonding for the formation of the aggregates decreases linearly with an increase in the peptide residue number. This relationship may apply to the aggregate systems of the LG3, LG4, and LG5 oligopepides.

5832 J. Phys. Chem. B, Vol. 112, No. 18, 2008

Yoshino et al.

The dimeric complexes of these L-leucyl-glycine oligopeptides in aqueous solution may be regarded as one-dimensional aggregates. Accordingly, the one-dimensional aggregate theory29 may be applied to these most simple aggregate systems to evaluate the energy of intermolecular hydrogen bonding for the formation of aggregates. In the one-dimensional model,29 we may define the bond energy (in this present study, the energy of intermolecular hydrogen bonding) between monomers in the aggregate, relative to isolated monomers in solution, as equal to RκT (κ is the Boltzmann constant, and T is the absolute temperature). The density distribution function (D) of molecules in an aggregate consisting of i molecules is provided by

Di ) i[1 - (1/xfeR)]ie-R/f

(7)

where f is the molal fraction of the solute. Eq 7 is restricted to dilute systems in which interactions between the aggregates can be ignored. Since the density function (Di) takes the maximum i value (imax) at ∂Di/∂i ) 0, imax is expressed as

imax ) xfeR ) Nn

(8)

where imax is equal to the number-average aggregation number (Nn). Thus, the bond energy RκT between monomers in the onedimensional aggregation model can be calculated from

RκT ) [ln(Nn2/f)]κT

(9)

implying that the intermolecular hydrogen-bond energy is determined by the Nn and f values at constant temperature. For the dimeric complexes of LG3, LG4, and LG5, the values of RκT and RκT/Np were calculated, based on the assumption that the interaggregate interactions were negligible. Values of RκT ) 14.78 kJ/mol (RκT/3 ) 4.93 kJ/mol) for LG3 (C ) 0.173 mol/L), RκT ) 15.14 kJ/mol (RκT/4 ) 3.79 kJ/mol) for LG4 (C ) 0.145 mol/ L), and RκT ) 16.14 kJ/mol (RκT/5 ) 3.23 kJ/mol) for LG5 (0.086 mol/L) were obtained. The energy of most hydrogen bonds is in the range of 1040 kJ/mol,29 which makes them stronger than a typical van der Waals force (below ca. 1 kJ/mol). The RκT values obtained for these dimeric aggregate systems are in this energy range, indicating that we may use the RκT values as an indicator of the energy of intermolecular hydrogen bonds required to form dimeric aggregates and are exactly equal to the hydrogen-bond energies. The energy values calculated are in the lower side of this range, indicating that these energies are relatively small. Figure 9 shows the plots of the RκT/Np value against the number of peptide residues (Np) for the L-leucylglycine oligomeric systems. It is evident that there is a linear relationship between RκT/Np and Np, showing that the energy of hydrogen bonds per peptide residue between the monomers in the dimeric aggregates decreases linearly with an increase in Np. The magnitude of the slope of a straight line in this plot may be used as an indicator of the variation in the energy of intermolecular hydrogen bonding due to the elongation of the oligopeptide through the addition of peptide residues. From the linear relationship in the plots, an absolute value of the slope equal to 0.85 kJ/mol per peptide residue was obtained. The extrapolation of the RκT/Np value to zero provides the number of peptide residues Np ) 8.7 at RκT/Np ) 0. This result indicates that the energy of intermolecular hydrogen bonds to

Figure 9. Plots of the monomer-monomer bond energies per peptide residue (RκT/Np) against the peptide residue number (Np) for the LG3-, LG4-, and LG5-D2O system (b) and for the ANpZ-dioxane(d8) system (2, cited from ref 12). The straight lines were obtained by linear leastsquares fits.

form the dimeric aggregate becomes very small (0.58 kJ/mol per peptide residue) or almost zero at Np ) 8 or 9, implying that the longer L-leucylglycine oligopeptides LG8 and LG9 do not form dimeric aggregates. Furthermore, values of RκT/Np equal to 2.28 and 1.43 kJ/mol are predicted for LG6 (Np ) 6) and LG7 (Np ) 7), respectively. To judge as to whether or not the existence of LG6 and LG7 is actually possible, plots of RκT/Np versus Np for the ANpZ (Np ) 6, 8, 10, 12, and 14)-dioxane system (cited from previous work12) also are shown in Figure 9 for comparison. For the ANpZ-dioxane system, clear evidence of the microstructures of their aggregates was confirmed by SANS and SAXS studies. The absolute value of the slope obtained from the straight line is 0.34 kJ/mol per peptide residue, and extrapolation of the RκT/ Np value to zero provides Np ) 20.2. The RκT/Np values calculated for LG3, LG4, and LG5 (Figure 9, filled circles) are in the energy range obtained for the ANpZdioxane system (Figure 9, filled triangles), indicating the validity of these energy values. In particular, the energy value (2.28 kJ/ mol per peptide residue) predicted for LG6 corresponds closely to that (2.1 kJ/mol per peptide residue) for ANpZ (Np ) 14) obtained from the plots. It has already been confirmed that ANpZ molecules with Np ) 14 form an aggregate with an aggregation number equal to 18 in dioxane (concentration of 1.1 × 10-2 mol/L).12 Accordingly, we may assume that the predicted RκT/ Np value (2.28 kJ/mol) is a valid measure for the formation of the dimeric aggregates of LG6. The absolute value (0.85 kJ/mol) of the slope of the straight line in the plots for the L-leucylglycine oligopeptides is greater than that (0.34 kJ/mol) for the ANpZ oligomers. This fact indicates that, for the L-leucylglycine oligopeptides, the reduction of the intermolecular hydrogen-bonding energy due to elongation of the oligopeptide through the addition of Gly residues is much greater in extent than that of the ANpZ system. Accordingly, elongation by only three more glycine residues renders an intermolecular hydrogen-bonding energy (and subsequent formation of dimeric aggregates) very small or almost zero, possibly inducing the transition from the folded structure to another conformation, for example, a helical structure similar to that of polyglycine II33 (indeed, the LG5 molecule takes up this conformation in the crystalline state32). For the ANpZ-dioxane system,12 we emphasize that the addition of six peptide residues to ANpZ (Np ) 14) makes the

Folded Structures of L-Leucylglycine Oligopeptides intermolecular hydrogen-bonding energy almost zero. This result implies that the contribution of intermolecular hydrogen bonding to the formation of the rod-like aggregate, in which the flat β-sheet oligomers are one-dimensionally stacked in an antiparallel manner, becomes very small or zero at a residue number of ca. 20. As a consequence, it was assumed that the β-sheet structure becomes unstable, resulting in a decrease in the RκT/ Np value and that any further increase in the chain length may induce a conformational transition. Thus, the one-dimensional aggregate theory29 predicts that for LG6, a folded structure and the formation of a dimeric aggregate may be possible, judging from the calculated value of RκT/Np (2.28 kJ/mol per peptide residue), but for Lleucylglycine oligopeptides longer than LG7, there is little possibility for a folded structure. Furthermore, as a consequence of analyses based on this model, it has been assumed that elongation through the addition of glycine residues brings about a marked reduction of the intermolecular hydrogen-bonding energy, probably causing the marked increase in the flexibility of the LG5 dimeric aggregate. Conversely, the lower segmental mobility of the LG3 and LG4 dimeric aggregates probably arises from the greater contribution of the intramolecular hydrogen bonds to stabilization of the folded structure. Conclusion In aqueous solutions of LG3, LG4, and LG5 above their cac, folded molecular conformations preferentially were stabilized to form dimeric aggregates. The one-dimensional aggregate theory was applied to these systems. The results indicated that the energy of intermolecular hydrogen bonding leading to the formation of dimeric aggregates decreases linearly with an increase in the glycine residue number. This linear relationship provides the following prediction. The longer oligopeptide LG6 molecules may form relatively stable dimeric aggregates through intermolecular hydrogen bonding. Further elongation of the oligopeptide through the addition of glycine residues up to LG7 or LG8 probably makes these dimeric aggregates very unstable, implying that there is little possibility for their existence. The longer LG9 molecules do not form dimeric aggregates. Thus, the one-dimensional aggregate theory may be used to predict the existence of peptide aggregates formed through intermolecular hydrogen bonding. References and Notes (1) Okabayashi, H.; Okuyama, M.; Kitagawa, T. Bull. Chem. Soc. Jpn. 1975, 48, 2264. (2) Okabayashi, H.; Abe, M. J. Phys. Chem. 1980, 84, 999. (3) Okabayashi, H.; Yoshida, T.; Ikeda, T.; Matsu-ura, H.; Kitagawa, T. J. Am. Chem. Soc. 1982, 104, 5399. (4) Tsukamoto, K.; Ohshima, K.; Taga, T.; Okabayashi, H.; Matsuura, H. J. Chem. Soc., Faraday Trans. 1 1987, 83, 789.

J. Phys. Chem. B, Vol. 112, No. 18, 2008 5833 (5) Okabayashi, H.; Tsukamoto, K.; Ohshima, K.; Taga, K.; Nishio, E. J. Chem. Soc., Faraday Trans. 1 1988, 84, 1639. (6) Takahashi, H.; Nakayama, Y.; Hori, H.; Kihara, K.; Okabayashi, H.; Okuyama, M. J. Colloid Interface Sci. 1976, 54, 102. (7) Okabayashi, H.; Ohshima, K.; Etori, H.; Taga, K.; Yoshida; Nishio, E. J. Phys. Chem. 1989, 93, 6638. (8) Okabayashi, H.; Taga, K.; Yoshida, T.; Ohshima, K.; Etori, H.; Uehara, T.; Nishio, E. Appl. Spectrosc. 1991, 45, 626. (9) Becker, E. L.; Bleich, H. E.; Day, A. R.; Frear, R. I.; Ghasel, J. A.; Vinsintainner, J. Biochemistry 1979, 16, 4656. (10) James, M. N. G.; Brayer, G. D.; Delbaire, L. T. T.-J.; Sielecki, A. R.; Gertler, A. J. Mol. Biol. 1980, 139, 423. (11) Okabayashi, H.; Ishida, M.; Tamaoki, H.; Masuda, H.; O’Connor, C. J. Biopolymers 2002, 65, 129. (12) Ishida, M.; Takai, M.; Okabayashi, H.; Masuda, H.; Furusaka, M.; O’Connor, C. J. Phys. Chem. Chem. Phys. 2001, 3, 3140. (13) Isenberg, I. Annu. ReV. Biochem. 1979, 48, 159. (14) Beaudette, N. V.; Fulmer, A. W.; Okabayashi, H.; Fasman, G. D. Biochemistry 1981, 20, 6526. (15) Levitt, M.; Chothia, C. Nature (London, U.K.) 1976, 261, 552. (16) Bandekar, J.; Krimm, S. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 774. (17) Venkatachalam, C. M. Biopolymers 1968, 6, 1425. (18) Lewis, P. N.; Momany, F. A.; Scheraga, H. A. Biochim. Biophys. Acta 1973, 303, 211. (19) Scheraga, H. A. Pure Appl. Chem. 1973, 36, 1. (20) Kotelchuck, D.; Scheraga, H. A. Proc. Natl. Acad. Sci. U.S.A. 1969, 62, 14. (21) Anfinsen, C. B.; Scheraga, H. A. AdV. Protein Chem. 1975, 29, 205. (22) Bode, K.; Goodman, M.; Mutter, M. HelV. Chim. Acta 1985, 68, 705. (23) Gierasch, L. M.; Deber, C. M.; Madison, V.; Niu, C.-H.; Blout, E. R. Biochemistry 1981, 20, 4730. (24) Seaton, B. A. Spectrochim. Acta, Part A 1986, 42, 227. (25) Ueki, T.; Ashida, M.; Kakudo, M.; Sasada, Y.; Katsube, Y. Nature (London, U.K.) 1967, 25, 1840. (26) Yagi, Y.; Tanaka, T.; Yamane, T.; Ashida, T. J. Am. Chem. Soc. 1983, 105, 1242. (27) Prange, T.; Sakarellos, C.; Toma, F.; Pascard, C.; Fermanjian, S. J. Am. Chem. Soc. 1983, 105, 6306. (28) Srikrishnan, T.; Parthasarathy, R. Int. J. Pept. Protein Res. 1987, 30, 557. (29) Israelachivili, N. J. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1991; Ch. 16, p 341. (30) Hashimoto, A.; Aoyagi, H.; Izumiya, N. Bull. Chem. Soc. Jpn. 1980, 53, 2926. (31) Bandekar, J. Biochim. Biophys. Acta 1992, 1120, 123. (32) Ohshima, K.; Okabayashi, H.; Yoshida, T. Vibr. Spectrosc. 1995, 8, 401. (33) Dwivedi, A. M.; Krimm, S. Macromolecules 1982, 15, 177. (34) Krimm, S.; Bandekar, J. Biopolymers 1980, 19, 1. (35) Jonsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants and Polymers in Aqueous Solution; John Wiley and Sons: Chichester, U.K., 1998; Ch. 2, p 38. (36) Beecham, A. F.; Ham, N. S. Tetrahedron 1968, 24, 2773. (37) Abragam, A. The Principles of Nuclear Magnetism; Oxford University Press: London, 1961; Ch. 8. (38) (a) Momany, F. A.; McGuire, R. F.; Burgess, A. W.; Scheraga, H. A. J. Phys. Chem. 1975, 79, 2361. (b) Beppu, Y. Comput. Chem. 1989, 13, 101. (c) Sisido, M. Pept. Chem. 1992, 105, 1991. (39) Srikrishnan, T.; Vniewicz, N.; Parthasarathy, R. Int. J. Pept. Protein Res. 1982, 19, 103.