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Keywords:

  • tetrapeptide;
  • WUGW peptide;
  • beta turns;
  • infrared absorption;
  • vibrational circular dichroism;
  • density functional predictions

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

The intramolecularly hydrogen bonded conformations of the tetrapeptide Boc-Trp-Aib-Gly-Trp-OMe (WUGW) are investigated using experimental and quantum chemical predictions of vibrational circular dichroism (VCD) in the 1800–1550 cm−1 region. The predicted VCD spectrum, for a conformation (conformer A) obtained from optimization of crystal structure, reproduced the dominant negative VCD band observed experimentally in CH3OH and CHCl3 solvents. However, the predicted VCD spectrum of Conformation A also has an extra positive band which is not seen in the experimental spectra. This mismatch appears to be due to the lack of solvent influence in the quantum chemical geometry optimizations. However, Conformations I and II, obtained, respectively, from constrained optimization of crystal and NMR structures, mimic the solvent stabilized structures and are predicted to have dominant negative VCD band as found in the experimental spectra. It is noted that, for the peptide investigated here, unconstrained quantum chemical geometry optimizations in vacuum converged to structures that are not the realistic models of conformations found in solution. It is also noted that undertaking quantum chemical vibrational property calculations directly using geometries obtained from crystal data or NMR data resulted in unrealistic vibrational frequencies and descriptions. However, constraining the backbone dihedral angles to those found in condensed medium, and optimizing the remaining geometrical parameters resulted in a better reproduction of the observed VCD in condensed medium. The vibrational origins of bands in all of the predicted VCD spectra for the WUGW-tetrapeptide have also been presented. Chirality 21:E76–E85, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

Structures of biomolecules are typically investigated using X-ray crystallography, nuclear magnetic resonance (NMR), and/or electronic circular dichroism (ECD), depending on the applicability of these individual methods. Recently, vibrational circular dichroism (VCD) spectroscopy has emerged as a powerful method for the structural elucidation of peptides and proteins in solution and film states. Although both ECD and VCD spectroscopies allow investigation of peptide and protein structures, the ECD spectral interpretations are sometimes ambiguous1–3 when aromatic residues are present. This is because aromatic amino acids (Phe, Tyr, Trp) have electronic transitions in the far UV-region (190–230 nm) that overlap1 with those of amide groups and, as a consequence, ECD associated with these transitions can interfere with each other. On the other hand, as the vibrational transitions of aromatic residues and amide groups are well separated in frequency positions, VCD associated with these transitions do not interfere with each other. Thus, VCD spectroscopy offers definite advantages for structural determination of peptides and proteins that contain aromatic residues. The tetrapeptide investigated here contains two aromatic residues.

A number of studies4–19 have used VCD spectroscopy to derive conformationally sensitive information for protein/peptide systems. The secondary structures are reflected in the recognizable VCD signatures associated with the amide I (C[DOUBLE BOND]O stretch) and amide II (N[BOND]H deformation) vibrations. Although a majority of structural elucidations of peptides/proteins using VCD spectroscopy have been accomplished with empirical spectra-structure correlations, some predictive calculations have also been attempted. Initial VCD predictions1, 20 have been based on extended coupled oscillator model,21 which assumes22 dipole–dipole coupling between amide groups of amino acid units as the source of the optical activity. Keiderling and coworkers have promoted23–31 correlations of the experimental VCD spectra with corresponding approximate quantum chemical spectra using a transfer parameter method. This method relies on transferring parameters (geometry, force constants, and atomic tensors) obtained for smaller peptides, typically di- or tri-peptides, to larger polypeptides or proteins. This approach was deemed to give satisfactory predicted-experimental spectral correlations, while avoiding the high computational cost of fully quantum chemical treatment for larger systems. Thismethod has been implemented for α-helical,25, 26 β-sheet,27, 28 and β-hairpin29–31 secondary structures. However, the transfer method does not address or verify fundamental rules such as the sum rules for force constants32 and atomic tensors33–35 following the transfer of these parameters. As a result it is not clear whether, or not, the transfer parameter method gives satisfactory predicted-experimental spectral correlations for the “right” reasons. Furthermore, for aromatic systems, such as the one considered here, the π–π interactions between aromatic residues can dominate and such interactions are not likely to be represented correctly in the transfer parameter method.

Quantum chemical optimizations and VCD predictions, without transferring parameters, have been performed on alanine-based decaamide,36, 37 with helical secondary structure, and alanine-based triamide38 with β-sheet arrangement. Such fully quantum chemical VCD predictions have not been undertaken for hetero-amino-acid peptides or peptides capable of forming β-hairpin and β-turn type structures. In the case of cyclosporin, a 11-member cyclic peptide containing β-turn and sheet structural components, quantum chemical predictions of VCD obtained for fragment models39 were used to interpret the experimental VCD spectra.

Among the secondary structures of peptides and proteins, turn geometry displays40–43 considerable conformational variability. Turn is the non-repeating area where a polypeptide chain reverses its overall direction. In β-turn, this area comprises four amino acid residues, while in γ-turns three residues. Beside β- and γ-turns, additional types of turns are also known to exist.40–43 If four residues comprising a β-turn are labeled consecutively as i, i+1, i+2, and i+3 then the values of φi+1, ψi+1, φi+2, ψi+2 angles define41–43 the type of β-turn. These angles, respectively, of −60°, −30°, 90°, and 0° define Type I, −60°, 120°, 80°, and 0° define Type II, and −60°, −30°, −60°, and −30° define Type III. The corresponding angles with opposite signs define prime turns, Type I′, II′, and III′, respectively (which are also called inverse turns). β-hairpins usually contain41 Type I′ or II′ β-turns. Unlike fairly well-established VCD signatures for α-helix, β-sheet, and random-coil secondary structures,23, 44 characteristic VCD features for peptides exhibiting a turn motif have not been well defined. Xie et al. have reported20 based on both experimental and theoretical considerations that cyclic-peptides display a positive couplet in the amide I region as a dominant signature of Type I or Type II β-turns. Specifically, Type I turn was associated with the couplet at ∼1675/1690 cm−1, while Type II turn was associated with a broader couplet at ∼1635/1690 cm−1. However, the positive VCD couplet in the amide I region can also be interpreted45 as indicative of a γ-turn for cyclic-peptides. A different study,46 performed by Wyssbrod et al., has suggested that for cyclic-peptides with at least four residues, β-turn characterizing VCD pattern in the amide I region is a negative-positive-negative signal. Experimentally and theoretically considered VCD spectra for turn-forming tetrapeptides, reported47 by Hilario et al., have displayed a negative VCD couplet in amide I region. It was also indicated in the literature46 that linear hexapeptides exhibit amide I VCD pattern that is more complex than just a single couplet. On the basis of the previous VCD studies,20, 45–48 there is no prototypical VCD signature for turn as a secondary structural motif. VCD spectra may be able to distinguish among various types of turn conformations, but to establish conformational details of these turn conformations it is necessary to simultaneously obtain and compare the experimental and theoretical VCD spectra for peptides that have propensity for turn conformations.

β-hairpin structures result from a mixture of β-sheet and turn motifs. β-hairpins generally yield1, 2 an intense negative VCD band in the ∼1645 cm−1 region and sometimes also a weak positive VCD band at ∼1690 cm−1. Parallel β-sheet structures typically give4–19 one negative VCD signal at about ∼1630 cm−1. If both β-sheet and hairpin structures yield negative VCD signals then turn structure may also be associated with a negative VCD signal or no VCD signal at all. But to establish the VCD signature of turn motifs it is necessary to investigate the peptides that have turn structure only and no other secondary structural motif.

Although quantum chemical VCD simulations for β-turn-forming tetrapeptides have been considered in the past,47 the tetrapeptides for which these simulations have been performed differ in residue-composition from the tetrapeptide used for experimental investigation. Specifically, experimentally investigated tetrapeptides were Ac-Val-Asn-Gly-Lys-NH2 and Ac-Val-Pro-Gly-Lys-NH2, while theoretical investigation were conducted for tetra peptides with all residues, other than Pro and Gly, substituted with Ala residue. The geometry used for the calculations on these β-turn models is not a quantum chemically optimized geometry, but a Type I′ turn geometry extracted from the NMR structure of a larger peptide.

More recently, Keiderling and coworkers49 have reported a modeling study to establish the expected VCD signatures for Type I, I′, II, II′, III, and III′ β turns using Ac-Aib-Gly-NMe, Ac-Ala-Ala-NMe, and Ac-DPro-Gly-NMe as model compounds. However, experimental data are not available for these model compounds to assess the validity of predictions obtained.

From the literature background provided in the previous paragraphs, two deficiencies are apparent: (a) experimental VCD spectra for a short peptide with β-turn as the sole structural motif are not available; (b) Fully quantum chemical predictions of VCD spectra for the same β-turn-forming peptide, as the one experimentally studied, have not been reported to date. These deficiencies are remedied in the current study by presenting the first combined experimental and quantum chemical investigation, including both geometry optimization and VCD spectral prediction, done on the entire tetrapeptide that is residue-for-residue equivalent to the tetrapeptide investigated experimentally.

The tetrapeptide under investigation is Boc-Trp1-Aib2-Gly3-Trp4-OMe (WUGW)50 and it falls under the class of small tetrapeptides that exhibit well defined folded structures. This sequence (WUGW) was chosen to examine the folding propensity of the Aib-Gly segment when flanked by bulky aromatic Trp residues. The Aib-Gly segment has been previously shown to have strong tendency to form β turn conformations with two residues occupying the (i+1) and (i+2) positions. The choice of the present sequence was intended to examine the role of aromatic interactions across the i and (i+3) positions. Earlier studies from this laboratory have examined aromatic interactions in both the helices51 and hairpins.52 The achiral nature of the Aib and Gly residues permits the central β turn to adopt either a Type I/III or Type I′/III′ β turn conformation. The prime turns facilitate registered anti-parallel hairpin conformations while Type I/III turns can nucleate helical folding.53, 54 For the current WUGW-tetrapeptide, X-ray and NMR studies50 establish the presence of folded β-turn conformation. ECD spectrum of this tetrapeptide in CH3OH is anomalous and direct interpretation of secondary structure was not possible50 from that spectrum. The reported50 X-ray and NMR studies on this tetrapeptide suggest the presence of folded reverse turn conformations. The quantum chemical predictions of stable conformations, and of conformation dependent VCD spectra, along with the vibrational origins of the dominant VCD signals, would be of significant importance for this tetrapeptide. The comparison of predicted VCD spectra for different conformers with the experimental VCD spectra for this tetrapeptide may permit identification of the characteristic VCD signatures for some of the turn-alone type structures.

EXPERIMENTAL

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

The vibrational absorption (VA) and VCD spectra were recorded in the 2000–900 cm−1 region using a commercial Fourier transform VCD spectrometer modified55 to reduce the level of artifacts. The VCD spectra were recorded with 3 h data collection time at 4 cm−1 resolution. The sample was held in demountable cell with CaF2 windows and a 100 μm spacer. Spectra were measured in CHCl3 and CH3OH solvents at a concentration of ∼2 mg in 100 μl. Baseline corrections for all spectra were made by subtracting the absorption (or VCD) of the corresponding solvent. Spectral region below ∼1550 cm−1 has been omitted due to the interference from the solvent absorption.

The quantum chemical calculations were done using Gaussian 03 program. Theoretical optimizations and VA and VCD predictions were done using B3LYP functional and 6-31G* basis set. Other functionals and larger basis sets were not explored due to the constraints of computing time. Lorentzian band shapes, with a bandwidth of 10 cm−1, were used for spectral simulation. Predicted frequencies associated with optimized conformations A and B and constraints-imposed partially optimized Conformations I and II have been scaled by a factor of 0.9613.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

The WUGW-tetrapeptide provides a good model for studying multiple well folded conformations in a short peptide. The central Aib-Gly segment is capable of adopting both Type I and I′ structures. The flanking Trp residues can influence conformational choice by aromatic-amide interactions. The crystal structure of the WUGW peptide establishes a consecutive β-turn conformation, while NMR results provided evidence for interconversion between two conformers with equal populations in solution, one of which is also observed in the X-ray analysis.50 In this report, we investigate the secondary structure of WUGW-tetrapeptide using combined experimentally observed and quantum chemically predicted VCD spectra.

The geometry optimization of WUGW peptide involved consideration of three starting structures (Conformations I–III as given in Fig. 1). Each of the three starting structures represents a folded, fairly compact conformation with two hydrogen bonds. Conformation I is based on the coordinates obtained from an X-ray diffraction study50 of WUGW peptide crystals. This initial conformation exhibits backbone folding and is classified as a consecutive Type II–I′ β-turn.50 Conformation II is one of the conformations suggested by the NMR resuts50 and its dihedral angles resemble the conventional Type I′ β-turn at the Aib-Gly segment. These two initial conformations are representatives of β-turn secondary structures and differ only in the φ dihedral angle of Trp1 residue (refer Table 1): this angle is ∼−55° for Conformation I and ∼−150° for Conformation II. Conformation III has been constructed to model a 310-helical structure, which is widely observed in Aib containing peptides.56–59 The main structural parameters of these three initial conformations are presented in Table 1. These parameters include standard backbone dihedral angles (φ, ψ, ω) associated with each of the four amino acids and dihedral angles (χ1, χ2) associated with the orientation of two Trp residues.

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Figure 1. Structures of initial, non-optimized conformations of WUGW-tetrapeptide (Conformations I, II, and III). Conformation I is taken from crystal structure (Ref.50); Conformation II is derived from NMR data (Ref.50); Conformation III has been built with backbone dihedral angles of 310 helical structure.

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Table 1. Backbone dihedral angles (degrees) in the three starting initial conformationsa of Boc-Trp1-Aib2-Gly3-Trp4-OMe (WUGW) tetrapeptide
 φψbωbχ1cχ2c
  • a

    Conformation I is taken from crystal structure (Ref.50); Conformation II is taken from NMR structure (Ref.50); Conformation III has been built with backbone dihedral angles of 310 helical structure.

  • b

    Standard definitions of φ, ψ, and ω are used. For Trp4, ψ and ω correspond to dihedral angles N[BOND]C[BOND]C[BOND]O(CH3) and C[BOND]C[BOND]O[BOND]C(H3), respectively.

  • c

    χ1 and χ2 correspond to dihedral angles Nboc[BOND]C[BOND]C[BOND]C(CNtrp) and (NbocC)C[BOND]C[BOND]C[BOND]Ntrp, respectively, for Trp1 and N[BOND]C[BOND]C[BOND]C(CNtrp) and (NC)C[BOND]C[BOND]C[BOND]Ntrp, respectively, for Trp4.

Conformation I
 Trp1−55131177−57131
 Aib26120175  
 Gly3944179  
 Trp4−155−17717771−74
Conformation II
 Trp1−150131177−57131
 Aib26120175  
 Gly3944179  
 Trp4−155−17717771−74
Conformation III
 Trp1−61−30177−57131
 Aib2−60−31175  
 Gly3−61−31179  
 Trp4−60−30a177a178−74

The intra-molecular hydrogen bonds are depicted in Figure 1. For Conformation I, one hydrogen bond (∼2.29 Å) is between Trp1 C[DOUBLE BOND]O and Trp4 N[BOND]H, and another hydrogen bond (∼1.85 Å) is between Boc-ester C[DOUBLE BOND]O and Gly3 N[BOND]H. For Conformation II, one hydrogen bond (∼2.29 Å) is between Trp1 C[DOUBLE BOND]O and Trp4 N[BOND]H, and another hydrogen bond (∼2.32 Å) is between Trp1 N[BOND]H and Trp4 C[DOUBLE BOND]O. For Conformation III, one hydrogen bond (∼2.25 Å) is between Trp1 C[DOUBLE BOND]O and Trp4 N[BOND]H, and another hydrogen bond (∼1.94 Å) is between Boc-ester C[DOUBLE BOND]O and Gly3 N[BOND]H.

The optimizations, in vacuum, of these three conformations at B3LYP/6-31G* level theory have led to their convergence into two structures that differ from each of the starting structures. The structural parameters of the fully optimized conformations are given in Table 2, while the structures themselves are displayed in Figure 2. Conformers I and II have converged into an intermediate structure (designated as Conformation A) with φ of Trp1 residue as −75°. This dihedral angle is in between the angles of −55° and −150° for the starting Conformations I and II, respectively. Additionally, while the hydrogen bonds between Trp1 C[DOUBLE BOND]O and Trp4 N[BOND]H from conformers I and II, Trp1 N[BOND]H and Trp4 C[DOUBLE BOND]O from conformer II, Boc-ester C[DOUBLE BOND]O and Gly3 N[BOND]H from conformer I are preserved in conformer A, an additional hydrogen bond (∼2.41 Å), between Boc-ester C[DOUBLE BOND]O and Aib2 N[BOND]H is present in conformer A. Conformation III has also been modified as a result of optimization into a structure designated as Conformation B. While one hydrogen bond between Boc-ester C[DOUBLE BOND]O and Gly3 N[BOND]H (2.02 Å) present in Conformation B is preserved from Conformation III, a second hydrogen bond between Aib2 C[DOUBLE BOND]O and Trp4 N[BOND]H (2.17 Å) is newly formed as a result of optimization.

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Figure 2. Structures of fully optimized conformations of WUGW-tetrapeptide (Conformations A and B) using B3LYP functional and 6-31G* basis set. A is the converged conformation from initial structure of Conformer I or II (Fig. 1). B is the converged conformation from initial structure of Conformer III (Fig. 1).

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Table 2. Backbone dihedral angles (degrees) in the two fully optimized conformationsa of Boc-Trp1-Aib2-Gly3-Trp4-OMe (WUGW) tetrapeptide
 φψbωbχ1cχ2c
  • a

    A is the converged conformation from initial structure of conformer I or II (Table 1). B is the converged conformation from initial structure of conformer III (Table 1).

  • b

    Standard definitions of φ, ψ, and ω are used. For Trp4, ψ and ω correspond to dihedral angles N[BOND]C[BOND]C[BOND]O(CH3) and C[BOND]C[BOND]O[BOND]C(H3), respectively.

  • c

    χ1 and χ2 correspond to dihedral angles Nboc[BOND]C[BOND]C[BOND]C(CNtrp) and (NbocC)C[BOND]C[BOND]C[BOND]Ntrp, respectively, for Trp1 and N[BOND]C[BOND]C[BOND]C(CNtrp) and (NC)C[BOND]C[BOND]C[BOND]Ntrp, respectively, for Trp4.

Conformation A
 Trp1−7599−168−58101
 Aib24938176  
 Gly3106−25−162  
 Trp4−134179a180b61−81
Conformation B
 Trp1−74−13169−62110
 Aib2−52−36179  
 Gly3−8279−172  
 Trp4−147−46a−179b−160−108

On the basis of the difference in predicted electronic energy values (ΔE = 6.12 kcal/mol), conformer A is significantly more stable than conformer B. Despite this large energy difference, both conformers have been subjected to VCD calculations to note the differences in theoretical spectra and evaluate the agreement with experimental spectra.

The comparison of experimental VA (bottom) and VCD (top) spectra obtained in CHCl3 and CH3OH solvents with the corresponding theoretical spectra of Conformations A and B is displayed in Figure 3. As can be seen from this figure, the dominant experimental VCD feature present in the amide I (C[DOUBLE BOND]O stretching) region is a negative band in both solvents (1666 cm−1 in CHCl3 and 1655 cm−1 in CH3OH). These negative VCD bands, for both solvents, are associated with the highest intensity absorption bands (1674 cm−1 in CHCl3 and in CH3OH 1666 cm−1) in the region considered. A negative VCD band in the ∼1648–1658 cm−1 range was also seen2 in longer analogous-peptide sequences with β-hairpin (β-sheet + β-turn) structures in chloroform solvent. This negative band appears2 (refer Fig. 3) at a slightly lower frequency (by about 10 cm−1) in methanol solvent. Since the present tetrapeptide does not have a β-sheet structural component but also gives a negative VCD signal in the amide I region at ∼1650–1660 cm−1, this negative signal can be interpreted as originating from the β-turn structure.

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Figure 3. Comparison of experimental and predicted vibrational absorption (lower panel) and VCD (upper panel) spectra. The predicted spectra are for the two converged optimized conformations (Conformations A and B) obtained using B3LYP functional and 6-31G* basis set.

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For the predicted spectra, the turn structure represented by conformer A displays the characteristic features as a negative VCD signal at 1679 cm−1 and a positive VCD signal at 1730 cm−1. The dominant negative VCD signal at 1679 cm−1 for Conformation A, correlates well in terms of position and intensity with the experimental negative VCD signals in both solvents. The predicted negative VCD signal originates from C[DOUBLE BOND]O stretching vibration of Trp1 (refer Table 3), coupled to C[DOUBLE BOND]O stretching vibrations of Boc-ester, Aib2 and Gly3, with a scaled frequency of 1681 cm−1. The intensity of this simulated negative VCD band is reduced by a neighboring vibration with positive VCD at a scaled frequency of 1691 cm−1, which originates from C[DOUBLE BOND]O stretching vibration of Gly3, coupled to C[DOUBLE BOND]O stretching vibrations of Trp1 and Aib2. The simulated positive VCD signal at ∼1730 cm−1 of conformer A doesn't have a corresponding experimental band for correlation. This positive VCD signal originates from two vibrations with scaled frequencies of 1700 cm−1 and 1729 cm−1 (Table 3). The origin of the 1700 cm−1 vibration is C[DOUBLE BOND]O stretching vibration of Aib2 with coupling to C[DOUBLE BOND]O stretching vibrations of Trp1 and Gly3. The origin of the 1729 cm−1 vibration is C[DOUBLE BOND]O stretching of Trp4.

Table 3. Predicted parameters associated with five vibrations giving rise to the dominant VCD signals of Conformation A
  • a

    The relative displacements are listed for carbonyl groups belonging to Boc, Trp1, Aib2, Gly3, and Trp4 residue to identify which residue(s) has the most pronounced C[DOUBLE BOND]O stretching for a given predicted vibration.

Simulated VCD bands (cm−1)(+), 1657(−), 1679(+), 1730
Unscaled freq. (cm−1)17251749175917691799
Scaled freq. (cm−1)16591681169117001729
Dipole strength (10−40 esu2 cm2)470.73476.91636.96679.52777.64
Rotational strength (10−44 esu2 cm2)110.23−476.46155.3661.28267.94
Carbonyl groupRelative displacementa
Boc-ester1.0472−0.15160.0430−0.08710.1022
Trp10.17340.9881−0.17330.3233−0.0056
Aib20.0277−0.29710.19261.07170.0297
Gly3−0.01380.21191.1165−0.1269−0.1045
Trp40.0874−0.0457−0.10040.0351−1.1764

The turn structure represented by conformer B displays very weak VCD signals. The predicted VCD spectrum of Conformation B doesn't have any satisfactory agreement with the experimental spectra. The most noticeable negative VCD signal of conformer B is present at 1764 cm−1 and it originates from C[DOUBLE BOND]O stretching vibration of Trp4 residue (Table 4). Even for a qualitative level comparison, this negative VCD signal is too far-removed to be correlated with the negative experimental signal.

Table 4. Predicted parameters associated with five vibrations giving rise to the dominant VCD signals of Conformation B
  • a

    The relative displacements are listed for carbonyl groups belonging to Boc, Trp1, Aib2, Gly3, and Trp4 residue to identify which residue(s) has the most pronounced C[DOUBLE BOND]O stretching for a given predicted vibration.

Simulated VCD bands (cm−1)(−), 1676(+), 1699(−), 1764
Unscaled freq. (cm−1)17431765177317771836
Scaled freq. (cm−1)16761696170417091765
Dipole strength (10−40 esu2 cm2)282.44607.07754.22433.09425.81
Rotational strength (10−44 esu2 cm2)−37.6422.4540.71−32.15−108.87
Carbonyl groupRelative displacementa
Boc-ester0.11170.04410.44910.9991−0.0075
Trp1−0.19751.13940.0459−0.0481−0.0192
Aib21.11080.16570.1394−0.1922−0.0091
Gly30.20380.0961−0.98150.42630.0000
Trp40.00790.02000.00870.00791.2649

Although positions of weaker VCD bands (positive band at 1657 cm−1 for Conformation A and negative couplet at 1699 and 1676 cm−1 for Conformation B) have been labeled in Figure 3, there are no corresponding experimental bands. The origins of these weaker predicted bands, nevertheless, have been identified based on data in Tables 3 and 4 as follows: the positive VCD signal at ∼1657 cm−1 of Conformation A originates from C[DOUBLE BOND]O stretching vibration of Boc-ester with some coupling to C[DOUBLE BOND]O stretching vibration of Trp1; the predicted negative VCD band at ∼1676 cm−1 for conformer B originates from C[DOUBLE BOND]O stretching vibration of Aib2 with coupling to C[DOUBLE BOND]O stretching vibrations of Trp1 and Gly3; the predicted positive VCD band at ∼1699 cm−1 originates from three vibrations with scaled frequencies of 1696 cm−1, 1704 cm−1, and 1709 cm−1 (Table 4). Vibrations at 1696 cm−1 and 1704 cm−1 contribute to the intensity of the positive band, while vibration at 1709 cm−1 with negative rotational strength diminishes the positive VCD band intensity. The origin of the 1696 cm−1 is C[DOUBLE BOND]O stretching vibration of Trp1 with coupling to the C[DOUBLE BOND]O stretching vibration of Aib2. The origin of the 1704 cm−1 vibration is C[DOUBLE BOND]O stretching vibration of Gly3 with coupling to the C[DOUBLE BOND]O stretching vibration of Boc-ester and Aib2. The origin of the 1709 cm−1 vibration is C[DOUBLE BOND]O stretching vibration of Boc-ester with coupling to C[DOUBLE BOND]O stretching vibrations of Aib2 and Gly3.

It is disconcerting to note that the predicted VCD spectrum for conformer A has a positive VCD band at 1730 cm−1 with significant intensity for which no corresponding experimental band is seen. A possible explanation for this mismatch is that the calculations are done for isolated molecules, while the experimental data are obtained in solution phase. The NMR spectral data for WUGW suggested50 the presence of two conformers in equilibrium, but the geometry optimizations of these two conformers in vacuum lead to a single conformer A. Then it is to be construed that the NMR data are indicating solvent mediated stability of conformers I and II, and therefore, VCD calculations have to be undertaken for these two conformers.

The solvent influence on geometry optimizations can be incorporated using approximate models such as polarizable continuum model (PCM),60 but our attempts to optimize the current tetrapeptide geometries using PCM model turned out to be unsuccessful. Explicit inclusion of solvent molecules61 is one way to overcome this problem, but such calculations increase the number of basis functions leading to computational limitations.

We have also undertaken the VCD calculations at the geometries of conformers I and II, as obtained from crystal structure and NMR data, without any geometry optimization, but this procedure led to unrealistic vibrational mode descriptions. This is because, bond lengths and angles obtained from the crystal structure and NMR data are often significantly different from those in optimized geometries. For example, C[DOUBLE BOND]O bond lengths in crystal structure are ∼1.5 A, which are more like single bond lengths and, as a result, the predicted carbonyl stretching vibrations appeared at much lower frequencies mixed with C[BOND]C and C[BOND]N stretching vibrations.

To overcome these problems, we chose to fix the backbone dihedral angle values of φ, ψ, and ω in conformers I and II to those found in crystal and NMR data and optimize the bond lengths and angles. Such constrained optimization resulted in realistic bond lengths and angles while preserving the turn geometries found in crystal and NMR data. The constrained optimization was allowed for 65 cycles of optimization. Although conformers I and II with constrained optimization were not found to be at the potential energy minimum (as indicated by one imaginary vibrational frequency), this can be expected as the solvent mediated stability is not built into the calculation. Nevertheless, the vibrational spectra predicted for these conformers may be expected to capture the dominant features that would have been obtained by including explicit solvent influence.

Comparison of the experimental spectra with those predicted for constraints-imposed partially optimized Conformations I and II is presented in Figure 4. The predicted VCD spectra now display one predominant negative VCD band which corresponds to the negative experimental VCD band observed in solution. Specifically, the experimental negative VCD band at ∼1650 cm−1 arising from the highest intensity absorption band seems to correspond to the predicted negative VCD band (at 1689 cm−1 for conformer I and 1684 cm−1 for conformer II) also arising from the highest intensity absorption bands. Although there are also weak positive VCD bands in the predicted spectra they are not the dominant ones. Since both constraints-imposed partially optimized Conformations I and II are predicted to have a dominant negative VCD signal, as found in the experimental VCD spectra, one can surmise that both Conformations I and II might be present in solution, as deduced from the NMR study.50

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Figure 4. Comparison of experimental and predicted vibrational absorption (lower panel) and VCD (upper panel) spectra. The predicted spectra are for the two constraints-imposed partially optimized β-turn conformations (Conformations I and II) obtained using B3LYP functional and 6-31G* basis set.

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The origins of the predicted bands for constraints-imposed partially optimized Conformations I and II are provided in Tables 5 and 6, respectively. The correlated negative band at ∼1689 cm−1 of constraints-imposed optimized Conformation I originates from two vibrations with scaled frequencies of 1685 cm−1 and 1690 cm−1. Vibration at 1685 cm−1 with positive rotational strength diminishes the intensity of vibration at 1690 cm−1 with negative rotational strength. The origin of 1685 cm−1 vibration is the C[DOUBLE BOND]O stretching of Boc-ester with coupling to C[DOUBLE BOND]O stretching vibration of Aib2 and Gly3. The origin of the 1690 cm−1 vibration is the C[DOUBLE BOND]O stretching of Gly3 with coupling to C[DOUBLE BOND]O stretching vibrations of Boc-ester and Trp1. In terms of the correlated negative VCD band at ∼1684 cm−1 of constraints-imposed optimized Conformation II, it originates from C[DOUBLE BOND]O stretching vibration of Trp1 with coupling to C[DOUBLE BOND]O stretching vibrations of Boc-ester, Aib2 and Gly3.

Table 5. Predicted parameters associated with vibrations giving rise to the VCD signals of constraints-imposed optimized Conformation I
  • a

    The relative displacements are listed for different vibrations to identify which group(s) exhibits the most pronounced stretching.

Simulated VCD bands (cm−1)(+), 1672(−), 1689(+), 1710(+), 1751
Unscaled freq. (cm−1)17431753175817781823
Scaled freq. (cm−1)16751685169017101752
Dipole strength (10−40 esu2 cm2)679.66769.46464.22488.74425.27
Rotational strength (10−44 esu2 cm2)110.6064.15−299.30133.6585.55
Carbonyl groupRelative displacementa
Boc-ester−0.3931−0.99040.3777−0.1760−0.0371
Trp10.06990.1061−0.2020−1.07700.1336
Aib2−1.05060.4049−0.1456−0.00740.0000
Gly3−0.0127−0.4316−1.08890.14760.0871
Trp4−0.0313−0.00950.12380.10351.2681
Table 6. Predicted parameters associated with vibrations giving rise to the VCD signals of constraints-imposed optimized Conformation II
  • a

    The relative displacements are listed for different vibrations to identify which group(s) exhibits the most pronounced stretching.

Simulated VCD bands (cm−1)(−), 1684(+), 1698(+), 1742
Unscaled freq. (cm−1)17541761176617911814
Scaled freq. (cm−1)16861693169717221744
Dipole strength (10−40 esu2 cm2)899.58654.91527.49494.40429.81
Rotational strength (10−44 esu2 cm2)−374.70217.6864.0848.76122.77
Carbonyl groupRelative displacementa
Boc-ester0.19530.06780.02861.1477−0.1689
Trp1−1.0248−0.2275−0.35620.22020.0904
Aib20.2321−1.13140.05700.0287−0.0191
Gly3−0.3391−0.01871.13680.04690.1109
Trp40.14450.0091−0.08440.14451.2450

The results presented in the previous paragraphs suggest that both Type II–I′ β-turn (conformer I) and Type I′ β-turn (conformer II) yield a negative amide I VCD signal as the dominant feature. A negative VCD, followed by positive VCD at a much higher frequency, are predicted for a structure intermediate (conformer A) between conformers I and II. Since the experimental VCD spectra for WUGW peptide show a single dominant negative amide I VCD band, the presence of conformers I and II for WUGW peptide is implicated, in agreement with the predictions50 from NMR data.

For general conclusions regarding what type of VCD signals may be associated with β-turn geometries, it is to be noted that the current WUGW-tetrapeptide is known50 to exist in equilibrium between two Type I′ turn structures (conformers I and II). The experimental VCD spectrum of this tetra peptide exhibits a dominant negative VCD band in the 1670–1650 cm−1 region. Quantum chemical predictions of VCD for conformers I and II, do yield a dominant negative VCD signal (Fig. 4) that correlates with the experimental VCD band. Thus, it appears that typical Type I′ turns can be associated with one dominant negative VCD band at about 1650 cm−1. However, some variations to this generalization can be expected depending on the composition of residues and the distortion of angles in Type I′ turn. Conformer A, representing a distorted Type I′ turn, where the angles for i+1 and i+2 residues differed from standard turn angles by up to ∼25°, yielded a significant positive VCD band on the higher wavenumber side of the negative VCD band. A predominantly negative VCD band, with a weak positive shoulder on the higher wavenumber side, was also predicted49 for Type I′ turn in Ac-Ala-Ala-NMe. Thus it appears that although Type I′ turn can be associated with a dominant negative VCD band at about 1650 cm−1, sometimes an additional positive VCD band on the higher wavenumber side cannot be ruled out. Synthesis of, and VCD investigations on, additional small peptides containing turn structure as the sole structural motif are expected to be useful for expanding on these conclusions.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

The first comparison of experimental VCD spectra with corresponding fully quantum chemical (both optimization and VCD) predictions has been performed for a β-turn-forming WUGW-tetrapeptide. This report additionally establishes the vibrational origins of the bands in all of the predicted VCD spectra for the tetrapeptide under investigation. The predicted VCD spectrum of fully optimized Conformation A results in the reproduction of the dominant negative VCD signal observed experimentally, but exhibits an extra positive band which is not seen in the experimental spectrum. This mismatch could be resulting from the lack of solvent influence in the quantum chemical geometry optimizations. However, Conformations I and II, which mimic the solvent stabilized structures, are predicted to have dominant negative VCD bands as found in the experimental spectra, indicating that both conformations might be present in solution. On the basis of the observations for WUGW-tetrapeptide, it is noted that quantum chemical geometry optimizations for peptides with β-turn propensity in vacuum are not advisable since they are likely to converge to structures that are not the realistic models of conformations found in solution. It is also not advisable to do quantum chemical vibrational property calculations directly on geometries obtained from crystal data or NMR data, as they result in unrealistic vibrational frequencies and descriptions. In the absence of explicit solvent influence on calculated geometries, constraining the backbone dihedral angles to those found in condensed medium, and optimizing the remaining geometrical parameters appears to provide satisfactory vibrational properties for β-turn-forming peptides in the condensed medium.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

Computational work utilized the NCSA IBM P690 and SGI Altix systems. Teaching assistantship from Vanderbilt University (to A.G.P.) is greatly appreciated. R.M. was supported by the award of a Senior Research Fellowship from the Council of Scientific and Industrial Research, India.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED