NMR Analysis of Cross Strand Aromatic Interactions in an 8 Residue

Article pubs.acs.org/JPCB

NMR Analysis of Cross Strand Aromatic Interactions in an 8 Residue Hairpin and a 14 Residue Three Stranded β‑Sheet Peptide Rajesh Sonti,†,§ Rajkishor Rai,† Srinivasarao Ragothama,§ and Padmanabhan Balaram*,† †

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India 560012 NMR Research Centre, Indian Institute of Science, Bangalore, India 560012

§

S Supporting Information *

ABSTRACT: Cross strand aromatic interactions between a facing pair of phenylalanine residues in antiparallel β-sheet structures have been probed using two structurally defined model peptides. The octapeptide Boc-LFVDPLPLFV-OMe (peptide 1) favors the β-hairpin conformation nucleated by the type II′ β-turn formed by the D Pro-LPro segment, placing Phe2 and Phe7 side chains in proximity. Two centrally positioned DPro-LPro segments facilitate the three stranded β-sheet formation in the 14 residue peptide BocLFVDPLPLFVADPLPLFV-OMe (peptide 2) in which the Phe2/ Phe7 orientations are similar to that in the octapeptide. The anticipated folded conformations of peptides 1 and 2 are established by the delineation of intramolecularly hydrogen bonded NH groups and by the observation of specific cross strand NOEs. The observation of ring current shifted aromatic protons is a diagnostic of close approach of the Phe2 and Phe7 side chains. Specific assignment of aromatic proton resonances using HSQC and HSQC-TOCSY methods allow an analysis of interproton NOEs between the spatially proximate aromatic rings. This approach facilitates specific assignments in systems containing multiple aromatic rings in spectra at natural abundance. Evidence is presented for a dynamic process which invokes a correlated conformational change about the Cα-Cβ(χ1) bond for the pair of interacting Phe residues. NMR results suggest that aromatic ring orientations observed in crystals are maintained in solution. Anomalous temperature dependence of ring current induced proton chemical shifts suggests that solvophobic effects may facilitate aromatic ring clustering in apolar solvents.

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OMe (peptide 1) and Boc-LFVDPLPLFVADPLPLFV-OMe (peptide 2). The β-hairpin conformation of 1 has been previously characterized in crystals31 and in solution.28 The hairpin conformation of 1 and the anticipated three stranded βsheet structure of 2 are schematically illustrated in Figure 1. The design of 2 is based on the previously demonstrated ability of sequences containing multiple DPro-Xxx segments to fold into multistranded β-sheet structure.16,32,33 In both the peptides, Phe2 and Phe7 occupy facing non-hydrogen bonding positions in an antiparallel β-sheet, bringing the two phenyl groups into close proximity. This feature has been recognized in an earlier study which focused on the aromatic ring current induced shift of Cδ,δ1 H2 proton of Phe7.34 In crystal structures, fixed orientations of the two interacting aromatic residues may be characterized. In contrast, in solution dynamic process involving aromatic ring flips about the Cβ-Cγ(χ2) bond and the possibility of multiple conformational states about Cα-Cβ(χ1) need to be considered. Analysis of NOEs involving aromatic protons requires residue specific assignments of the ring protons. We describe the characterization of the three stranded

INTRODUCTION The tertiary structure of proteins involves interactions between side chains of amino acid residues which may be widely separated in the primary sequence. β-Hairpin structures of polypeptides provide an opportunity to study side chain interactions because of the spatial proximity of residues at nonhydrogen bonding sites. Aromatic interactions have been invoked as a stabilizing factor in the formation of isolated hairpin structures in solution.1−8 Model systems for the study of side chain interactions can be readily constructed using synthetic peptide β-hairpins as templates.9 Peptide hairpin design has been facilitated by the use of DPro-Xxx segments for nucleating prime turn formation, followed by antiparallel registry of β-strand segments.10−12 The efficacy of this design principle has been demonstrated by NMR studies in solution13−21 and by X-ray diffraction in crystals.11,22−28 The D Pro-LPro segment is a particularly effective hairpin nucleator, with the heterochiral dipeptide being an obligatory type II′ βturn forming unit.28−30 As part of a program to investigate orientations of aromatic rings at facing positions at nonhydrogen bonding sites in synthetic hairpins, we describe NMR studies on a model 8 residue hairpin and a designed 14 residue three stranded β-sheet generated by extension of the octapeptide. The sequences studied are Boc-LFVDPLPLFV© 2012 American Chemical Society

Received: April 11, 2012 Revised: July 13, 2012 Published: November 19, 2012 14207

dx.doi.org/10.1021/jp3034769 | J. Phys. Chem. B 2012, 116, 14207−14215

The Journal of Physical Chemistry B

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The homogeneity of the HPLC purified peptide was ascertained by reverse phase HPLC on a C18 column (5−10 μ). The purified peptide was analyzed by mass spectrometry on a Kratos Analytical (UK) Kompact SEQ MALDI-TOF mass spectrometer. MNa+obs 1692 (Mcalc = 1669)]. NMR Spectroscopy. NMR experiments were carried out on Bruker Avance 500 and 700 MHz spectrometers. NMR spectra were recorded at a peptide concentration of ∼3 mM in CDCl3 and CD3OH at 300 K. Delineation of exposed NH groups was achieved by titrating CDCl3 solutions with low concentrations of DMSO-d6. In the case of CD3OH, intramolecular hydrogen bonding was probed by recording 1D spectrum at five different temperatures ranging from 278 to 318 at 10 K intervals and determining the temperature coefficients of amide proton chemical shifts.35 Residue specific assignments were obtained from COSY36 and TOCSY37 experiments, while ROESY38 spectra permitted sequence specific assignments. Unambiguous aromatic proton and carbon chemical shifts were assigned by using a combination of 2D HSQC39 and HSQC-TOCSY40 experiments. All 2D experiments were recorded in phase sensitive mode using STATES-TPPI for TOCSY and ROESY and EchoAntiecho mode for HSQC and HSQC-TOCSY in the F1 dimension. A data set of 2048 × 400 was used for acquiring the data. The same data set was zero filled to yield a data matrix of size 4096 × 1024 before Fourier transformation. A spectral width of 6009 Hz was used in both dimensions for TOCSY and ROESY experiments. Mixing times of 100 ms for TOCSY, 50 and 250 ms for ROESY, and 80 ms for HSQC-TOCSY were used. Shifted square sine bell windows were used while processing. 1D homodecoupled spectra (NH protons) were acquired in order to permit specific assignment of the ABX spin systems of Phe2, Phe7, and Phe13 in order to extract the vicinal 3 α JC H‑CβH coupling constants. All processing was done using Bruker TopSpin 2.1 software. The spectra were analyzed using XEASY software.41 Structure Calculations. Structure calculations for peptide 2 were performed using CYANA 3.0 software.42,43 NOEs were classified as strong, medium, and weak by visual inspection, and the upper distance limits used are 2.5 Å, 3.5 Å, and 5.0 Å, respectively. Hydrogen bond constraints obtained from DMSO titration data in CDCl3 were used. A total of 94 distance restraints were used for structure calculations. Ten dihedral

Figure 1. Schematic representation of peptides 1 and 2. Expected hydrogen bonds are shown as broken lines.

β-sheet conformation of peptide 2, present the results of HSQC and HSQC-TOCSY experiments which permit the detailed assignment of aromatic protons, and correlate observed NOEs to multiple conformational states of the two phenyl residues in 1 and three phenyl residues in 2.

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MATERIALS AND METHODS Sample Preparation. Peptide 1 has been previously described.34 Peptide 2 was synthesized by conventional solution phase methods, using a fragment condensation strategy. The tert-butyloxycarbonyl (Boc) group was used for N-terminal protection, while the C-terminus was protected as a methyl ester. Deprotections were performed using 98% formic acid and saponification for N- and C-terminus, respectively. Couplings were mediated by dicyclohexylcarbodiimide/1hydroxybenzotriazole (DCC/HOBt). All the intermediates were used directly without further purification. The final peptide was purified by reverse phase, medium pressure liquid chromatography (C18, 40−60 μ) and by high performance liquid chromatography (HPLC) on a reverse phase C18 column (5−10 μ, 7.8 mm × 250 mm) using methanol−water gradients.

Table 1. NMR Parameters for Peptide 2 in CDCl3 and CD3OH Solutionsa Chemical Shift (ppm)

a

Residue

NH

Cα H

Leu(1) Phe(2) Val(3) D Pro(4) L Pro(5) Leu(6) Phe(7) Val(8) Ala(9) D Pro(10) L Pro(11) Leu (12) Phe(13) Val(14)

6.17 (6.99,6.57∧) 6.52(8.09,7.93∧) 8.83(8.96,8.80) ------7.66(7.96) 7.74(8.16) 8.20(8.33) 8.41 (8.45) ------7.45(7.68) 6.57(8.26) 8.65(8.69)

4.21 (4.25) 5.49 (5.40) 4.44 (4.47) 4.56(4.68) 4.70(4.61) 4.71 (4.72) 5.11(5.10) 4.47 (4.35) 4.78(4.8) 4.53(4.58) 4.59(4.48) 4.38(4.51) 5.39(5.06) 4.51 (4.40)

JNH‑CαH

dδ/dT (ppb/k)*

Δδb(ppm)

9.2 (8.8*) 9.3 (9.2) 9.6(9.6) ------8.9(8.9) 9.3(8.8) 9.5(9.1) 8.9(9.3) ------8.8 (9.0) 8.9 (8.9) 8.1(9.2)

**** (9.42) (3.82) ------(2.26) (5.9) (5.1) (5.92) ------(1.05) (7.40) (7.12)

0.34 1.23 −0.15

3

0 0 0.01 −0.21

−0.03 1.22 −0.27

The values in parentheses correspond to those in CD3OH solution. bΔδ = δ (CDCl3 + DMSO-d6) − δ (CDCl3). 14208



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rendering the Cδ,δ′ H2 and Cε,ε′ H2 protons equivalent on the NMR time scale. The utility of the HSQC and HSQC-TOCSY experiments rests on the fact that if chemical shifts of any of three sets of proton and carbon resonances appear distinct from those of the other residues, connectivity patterns can be readily traced. Some features of the chemical shift dispersion of the aromatic resonances are noteworthy. The Cδ,δ′ H2 protons of all three Phe residues in the three stranded β-sheet peptide 2 have widely different chemical shifts (7.28, 6.64, 7.08), with the Phe 7 Cδ,δ′ H2 protons appearing at the highest field. Upfield shifts of the Phe 2ζ, Phe 7ε, and Phe 7ζ protons and a small shift to lower field of the Phe 2δ are also evident in the spectrum of peptide 2. Backbone Conformation of Peptides. The β-hairpin conformation of the octapeptide 1 has been previously established in solution by NMR28 and in crystals by X-ray diffraction. In peptide 2, the sequence has been extended to construct a three stranded β-sheet structure, generated by chain reversals at two centrally positioned DPro-LPro segments (Figure 1). The wide dispersion of NH and CαH chemical shifts in the 14 residue peptide 2 (Table 1, Figures S1 and S2) in both chloroform and methanol is clearly indicative of a highly structured backbone conformation. The delineation of intramolecularly hydrogen-bonded NH groups was carried out in methanol by determination of temperature coefficients of NH chemical shifts measured over the range 278−318 K (Figure S6). The dδ/dT values are summarized in Table 1. Hydrogen/deuterium exchange experiments were carried out by dissolving the peptide in CD3OD and measuring the intensities of NH resonances as a function of time (Figure S7). The solvent exposure of the NH groups in chloroform was probed by determining changes in chemical shifts upon addition of a strongly hydrogen-bonding solvent, DMSO. The solvent titration curves are shown in Figure S8, and Δδ(δ (CDCl3 + DMSO-d6) − (CDCl3)) values are listed in Table 1. In an ideal three stranded β-sheet structure for peptide 2, as schematically illustrated in Figure 1, only two NH groups, Phe 2 and Phe13, are expected to be solvent exposed. Inspection of the data in Table 1 does indeed establish that these two resonances show the highest dδ/dT values in methanol (9.42, 7.40 ppb/K) and the highest Δδ values on titrating CDCl3 with DMSO (1.23 and 1.22 ppm). In addition, both Phe2 and Phe13 exhibit high rates of H/D exchange (Figure S7) in CD3OD. In the three stranded β-sheet 8 NH groups are expected to be internally hydrogen bonded, Leu1, Val3, Leu6, Val8, Ala9, Leu12, Val14, and Phe7 in CDCl3. Upon addition of low concentrations of DMSO, all these resonances exhibit low Δδ values supporting their involvement in intramolecular hydrogen bonding. In methanol, with the exception of Val14 NH (dδ/dT 7.13 ppb/K) the remaining 7 NH resonances have moderate or low temperature coefficients (5.09−1.05 ppb/K). The precise determination of the dδ/dT value of Leu1 was not achieved because of its close overlap with aromatic resonances. Four NH resonances Val3, Ala9, Val8, and Leu6 show significantly slower H/D exchange rates in methanol, consistent with involvement in cross strand hydrogen bonding. The Val14 NH shows a high dδ/dT value and also exchanges relatively rapidly, suggesting that fraying of the structure at the C-terminus is possible in methanol solution.24 It is pertinent to know that a minor species is indeed observed in CD3OH corresponding to a cis conformer, presumably about Ala9 and DPro10. Nuclear Overhauser Effects. Figure 3 shows partial ROESY spectra of peptide 2 in CDCl3, illustrating key NOEs. All the

angle restraints, which were obtained from 3JNH‑CαH coupling constants, were also used. The best 20 structures out of the calculated 100 structures were selected and superposed using PYMOL.44

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RESULTS Assignment of Resonances. Backbone and Side Chain Resonances. NMR studies for 2 were carried out in chloroform (CDCl3) and methanol (CD3OH) solutions. Residue specific assignments of backbone and side chain proton resonances, with the exception of aromatic resonances, were achieved using a combination of TOCSY and ROESY experiments. Fully assigned 1H NMR spectra in CDCl3 and CD3OH are shown in Supporting Information Figures S1 and S2. In chloroform, only a single species was observed which was subsequently established as the conformation in which all the Xxx-Pro bonds were trans. In methanol, minor conformations in slow exchange were detectable, but the major species was assigned to an all trans structure using diagnostic DPro CαH- LPro CδH NOEs (vide inf ra). Table 1 lists the chemical shifts of the backbone and selected side chain protons and 3JNH‑CαH values in peptide 1. A full listing of the chemical shifts are provided in the Supporting Information Table ST1. Inspection of Table 1 reveals that 3JNH‑CαH values lie between 8.1 and 9.6 Hz in chloroform and 8.8−9.6 Hz in methanol. Residues expected to adopt extended strand conformations in β-sheet structures are characterized by high 3JNH‑CαH values greater than 8.0 Hz. Aromatic Resonances. The Cδ,δ′ H2 protons of three aromatic rings can be specifically assigned by virtue of the NOEs to Cβ H2 protons of the Phe residues as illustrated in Supporting Information Figure S3 for peptide 2. A combination of HSQC and HSQC-TOCSY experiments permits specific assignments of peptide 2 as illustrated in Figure 2 and for

Figure 2. Partial a) HSQC and b) HSQC-TOCSY spectra of peptide 2 in CDCl3 illustrating the assignment of aromatic proton and carbon resonances. Connectivities of aromatic resonances are shown as broken lines.

peptide 1 in Supporting Information Figure S4. Cross sections along the F2 dimension for the phenyl Cε resonances in the 2D HSQC-TOCSY spectrum for peptide 2 are shown in Supporting Information Figure S5. A notable feature of the aromatic resonances in peptides 1 and 2 are the significant upfield shifts observed for specific resonances. The upfield shifts of the Phe 7 Cδ,δ′ H2 protons in 1 have been previously ascribed to the shielding resulting from the ring current of the proximal Phe2 residue.34 It should be noted that in these small peptides rapid ring flipping occurs about the Cβ-Cγ(χ2) bond, 14209



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Figure 3. Partial ROESY spectra of peptide 2 in CDCl3 highlighting (a) NH ↔ NH and (b) CαH ↔ CαH NOEs.

interstrand NOEs diagnostic of the three stranded β-sheet structure; dαiαj NOEs: Phe(2α)/Phe(7α), Val(8α)/Phe(13α) and dNiNj NOEs: Val(3)/Leu(6), Phe(7)/Val(14), Ala(9)/ Leu(12), and Leu(1)/Val(8) are intense and clearly observed. The two type II1 β turns were confirmed by the observation of key NOEs: DPro(4α)/LPro(5δ) and DPro(10α)/LPro11δ. An important long-range NOE is observed between the methyl group of the C-terminal ester function and Leu 6α, suggesting that strand registry is maintained in a significant population of conformations. In methanol, additional minor conformers are observed due to cis/trans isomerization about Xxx-Pro bonds. The major species corresponds to the all trans peptide backbone. An analysis of ROESY spectra of this species in methanol confirms the presence of all the interstrand NOEs diagnostic of a three stranded hairpin conformation (see Supporting Information Figure S9). Structure calculations were carried out using NOE derived distance restraints and the corresponding violations are listed in Supporting Information Table ST2. A total of 31 restraints involving backbone protons and 63 restraints using side chainbackbone NOEs were obtained from the experimental spectrum. In addition, hydrogen bond constraints (H−N distance ≤2.20 Å, O−N distance ≤3.20 Å as upper limits) derived from temperature and solvent dependent chemical shifts together with dihedral angles (φ) (120° ± 30°) obtained from 3JNH‑CαH vicinal coupling constants were used. Figure 4 shows the superposition of 20 structures which satisfy experimental restraints with a final backbone RMSD of 0.21 ± 0.08 Å. The dihedral angles are listed in Supporting Information Table ST3. These structure calculations were performed using the pseudoatom model to describe the side chain-backbone NOEs involving three phenyl rings at positions 2, 7, and 13. Structure calculations using only backbone proton NOEs yielded an ensemble of structures very similar to those illustrated in Figure 4. The listing of the backbone dihedral angles is provided in Supporting Information Table ST4. The NMR results clearly support the formation of a three stranded antiparallel β-sheet structure facilitated by the type II′ β-turn conformations at the two centrally positioned heterochiral diproline segments (residues 4−5 and 10−11). Cross Strand Interactions of Aromatic Side Chains. Interactions between spatially proximate aromatic rings have been implicated as important determinants of protein secondary structures.1 Several analyses of protein crystal

Figure 4. Superposition of the 20 best structures calculated for peptide 2 using NOE constraints from the ROESY spectra in CDCl3. Mean backbone rmsd: 0.21 ± 0.08 Å. Mean global heavy atom rmsd: 0.72 ± 0.16 Å.

structure data have pointed out the importance of the orientation between two closely packed phenyl rings. Burley and Petsko45,46 pointed out over twenty years ago that interplanar angles of approximately 0° (parallel) and 90° (perpendicular) were favored in proteins.47 Theoretical calculations of benzene dimers have also supported specific orientational preferences.48−55 In the case of Phe rings positioned at the non-hydrogen bonding sites in an antiparallel β-sheet, the two Phe rings can indeed approach one another very closely. In an earlier analysis, we have correlated the crystallographically observed orientations in a series of model octapeptide hairpins containing Phe residues at positions 2 and 7 with ring current induced shifts of aromatic proton resonances.34 Figure 5 compares the chemical shifts of the aromatic proton resonances in peptide 2 with those in the tripeptide Boc-LFV-OMe and two previously characterized octapeptide hairpins (Boc-LVV-DPG-LFV-OME and peptide 1; Boc-LFV-DPLP-LFV-OME). The corresponding chemical shifts of aromatic proton resonances are shown in Supporting Information Table ST5. In the isolated tripeptide fragment the aromatic proton resonances are closely clustered between 7.2 and 7.3 ppm. A slightly greater dispersion of the aromatic resonances is observed in the octapeptide Boc-LVV-DPG-LFVOME. In this case, shielding of the Phe 7δ and 7ζ protons is 14210



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stranded β-sheet 2, an even greater dispersion of the aromatic resonance is observed with the Phe 7 Cδ,δ′ H2 protons appearing at 6.64 ppm. The residue specific assignment of the three sets of aromatic proton resonances (Figure 2) facilitates a further consideration of the aromatic ring orientations in peptide 2. Figure 6 provides a comparison of the side chain-backbone and side chain-side chain NOEs involving residues Phe2 and Phe7 in the octapeptide hairpin 1 and the three stranded βsheet peptide 2. The observed interproton distances between the two proximate residues Phe2 and Phe7 observed in the crystal structure of the octapeptide 1 are also shown. The Phe 2 CδH/Phe 2 CεH ↔ Phe 7 CαH and Phe 2 CδH/Phe 2 CεH ↔ Phe 7 CδH NOEs are observed, suggesting that interring orientations in solution are close to that observed in the crystal structure. These NOEs are also observed in the case of the 14 residue peptide 2. In addition, the Phe 2 CαH ↔ Phe 7 CδH appears more intense in peptide 2 as compared to the octapeptide. In both cases, the Phe 2 CδH ↔ Phe 7 CδH NOE is observed. The results presented thus far suggest that in both the 8 and 14 residue peptides the phenyl rings of Phe2 and Phe7 are oriented in a manner similar to that observed in the crystal structure of the octapeptide 1. Several examples of octapeptides containing Phe residues at positions 2 and 7 have been crystallographically characterized in this laboratory. As many as 32 independent molecules from hairpin structures are available permitting a detailed analysis of aromatic ring orientations.31 Interestingly, in 31 examples, the two aromatic rings are oriented in a manner similar to that observed in the octapeptide 1. Interestingly, in the case of the peptide BocLFV-DPA-LFV-OMe (CCDC 821274), two different orientations of the Phe2/Phe7 pair are observed in two crystallographically independent molecules (Figure 7). One of these corresponds to the generally observed conformation in all the hairpins with the following side chain torsion angles: Phe2: χ1= −64.7 and χ2 = 86.9 and Phe7: χ1 = −177.2 and χ2 = 68.7 (conformer I). In the other molecule (conformer II), the pair of

Figure 5. Partial 500 MHz 1D 1H NMR spectra highlighting the aromatic proton resonances of (a) peptide 2, (b) peptide 1, (c) BocLVV-DPG-LFV-OME, and (d) Boc-LFV-OMe in CDCl3. Aromatic ring orientations in crystals are shown in the figure.31

evident since there is only one aromatic ring in the molecule. The observed shielding must result from the magnetic anisotropy of the flanking peptide units.56 Indeed, inspection of the crystallographically determined orientation of the Phe7 side chain places one of the Cδ,δ′ H2 protons directly above the plane of the Phe7 and Val8 peptide bond. Rapid flipping of the phenyl group about the Cβ-Cγ(χ2) bond renders the Cδ,δ′ H2 protons equivalent on the NMR time scale.57,58 A more dramatic upfield shift of the Phe 7 Cδ,δ′ H2 protons is observed in the octapeptide Boc-LFV-DPLP-LFV-OME (Figure 5b). In this octapeptide hairpin, the phenyl rings at positions 2 and 7 are closely packed with Phe 7 Cδ,δ′ H2 protons being positioned as close as 3.2 Å from the center of the phenyl ring in position 2 (CCDC 821276). The significant shift of the Phe 7 Cδ,δ′ H2 protons (6.71 ppm) is strongly suggestive that the orientation observed in crystals are maintained in solution. In the three

Figure 6. (a) The interresidue interproton distances between Phe(2) and Phe(7) side chains in crystals of peptide 1.31 Partial ROESY spectra of (b) peptide 1 and (c) peptide 2 in CDCl3. 14211



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indeed stabilizing, even in solution in a nonaqueous solvent like CDCl3. In both states, the Phe 7 Cδ,δ′ H2 protons is shifted to high field by the aromatic ring current of Phe2. Figure 9 shows the effect of lowering the temperature on the chemical shifts of the Phe 7 Cδ,δ′ H2 and Phe 7 CαH protons. In the range 300− 240 K, there is a small shift to higher field. At temperatures below 240 K, there is a pronounced shift to lower field of both sets of resonances. The initial upfield shift of the Phe 7 Cδ,δ′ H2 may be rationalized as arising from a small increase in the population of the states with the appropriate Phe orientations. The subsequent downfield shift upon further lowering the temperature merits comment. An intriguing possibility that may be considered is that at lower temperatures, the two aromatic side chains favor conformations which place the phenyl rings further away from one another and expose them to solvation. Solvophobic effects have indeed been invoked in accounting for association of large apolar surfaces in organic solvents.59−62 By analogy with the hydrophobic effect, solvophobic interactions may be expected to have a negative temperature coefficient. Another interesting feature is the broadening of Phe 7 Cα H proton between 260 and 230 K with a slight narrowing at lower temperatures. This observation is consistent with the occurrence of conformational exchange processes. Further evidence for conformational averaging about χ1 for the Phe2 and Phe7 residues in the 14 residue peptide 2 was obtained from analysis of 3JCαH‑CβH coupling constants. The transitions of ABX spin systems of Phe2, Phe7, and Phe13 were identified following decoupling of the respective NH proton resonances allowing extraction of relevant coupling constants. The experimentally determined values (Hz) are Phe2 = 3.5/8.6, Phe7 = ∼5.7/6.2, and Phe13 = 6.2/5.2 which are consistent with dynamic conformational averaging. Thus while the backbone conformation of the three stranded β-sheet peptide is well-defined, precise determination of the orientation of the Phe2 and Phe7 rings is not possible because of conformational dynamics.

Figure 7. Schematic representation of different conformers in crystals of Boc-LFV-DPA-LFV-OME.31

Phe residues are oriented differently with the following torsion angles: Phe2: χ1 = 178.5 and χ2 = 55.3 and Phe7: χ1 = −64.0 and χ2 = 90.9. In conformer I, the two aromatic rings point toward the N and C termini, while in conformer II, both rings are oriented toward the turn segment. The parameters defining the aromatic orientations in conformer I are as follows: Rcen = 5.13 Å, Rclo= 3.72 Å, and γ = 61.3°. In conformer II these are as follows: Rcen = 4.77 Å, Rclo= 3.55 Å, and γ = 24.6°; where Rcen is the centroid to centroid distance; Rclo is the shortest distance between carbon atoms on the two phenyl rings; and γ is the interplanar angle. It is clear that the aromatic interaction is maintained in both conformations. The process of interconversion involves changes in the χ1 angle at both the residues. In order to assess whether rapid conformational interconversion between the two conformational states with different aromatic orientation in solution, we turn to a closer inspection of the observed NOEs. Figure 8 shows partial ROESY spectra

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DISCUSSION The use of a centrally positioned DPro-LPro segment permits the construction of short peptides adopting well-defined βhairpin conformations. Multistranded antiparallel sheets can be generated by incorporation of multiple DPro-LPro segments, as exemplified in previous studies. β-Hairpins and multistranded structures may be used as templates to examine cross strand aromatic interactions. In the case of antiparallel strands the placement of phenylalanine residues at facing non-hydrogen bonding positions results in the phenyl rings being approaching one another within as interacting distance in crystals. The maintenance of this orientation in solution is apparent from the observation of ring current shifts for specific aromatic proton resonances, together with cross strand side chain NOEs. The NMR evidence presented here for the 8 residue (peptide 1) and the 14 residue (peptide 2) provides strong support for the anticipated secondary structures and the proximity of the phenyl rings of the Phe side chains at positions 2 and 7. Independently determined crystal structures provide evidence for two distinct conformers in which the cross strand aromatic interactions are maintained even though the orientations with respect to the hairpin backbone are dramatically different. Interconversions between these conformational states involve changes in the χ1 value at the two residues with the Phe2/Phe7 pair switching from g−/t orientation in conformer I to a t/g− orientation in conformer II. Table 2 lists key NOEs which may

Figure 8. Partial ROESY spectrum highlighting the aromatic side chains and backbone NOEs of (a) peptide 2 and (b) peptide 1 in CDCl3.

illustrating observed NOEs between the aromatic protons and the backbone and the side chain protons of neighboring residues. In the case of rapid exchange between two distinct conformations, NOEs characteristic of the individual structural states may be observed. Table 2 lists key observed NOEs which are diagnostic of conformer I or conformer II. It is clear that the NOEs listed in Table 2 provide evidence for the population of both conformational states in solution. The maintenance of the close approach of the two phenyl rings in both the conformations suggests that the aromatic interactions are 14212



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Table 2. Characteristic Conformational Dependent NOEs and Interproton Distances in Specific Conformationsa

a

The interproton distances (Å) indicated for conformer I and conformer II correspond to the distances for the closest proton pairs and not pseudoatom.31

Figure 9. (a) Distances of Phe 7δ and Phe 7α protons to the centroid of Phe2 ring are shown as double edged dotted arrows in the crystal structure of peptide 1.31 (b) Temperature dependence of Phe 7δ and Phe 7α protons of peptide 2 in CDCl3.

be diagnostic of the two conformational states in peptides 1 and 2. The corresponding interproton distances determined using the crystallographically determined coordinates for the two conformations are also summarized. In peptides of this size, weak NOEs can be detected for pairs of protons which are approximately 4.0 Å apart, with increasing cross peak intensity at shorter distances. The NOE data shown in Figure 8 and summarized in Table 2 establish that the observed NOEs are indeed incompatible with the presence of a single conformer in

solution. Indeed, NOEs characteristic of both conformers I and II are observed. The question that might arise is whether such a conformational transition in solution involves a correlated motion of both side chains or if intermediate states in which the aromatic interactions are disrupted are formed. Figure 10 schematically illustrates the pathways of interconversion that may be considered. The NMR data presented here does not permit a distinction between a pathway which involves the correlated 14213



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that the aromatic interaction is driven by solvent forces in organic solvents. Such solvophobic effects in which solvent entropy contributes to the clustering of the nonpolar surfaces in organic media has been referred to as the solvophobic effect in analogy with the well-known hydrophobic effect in aqueous medium.59−62 The present study suggests that further insights into clustering of aromatic residues across antiparallel strands may be derived by appropriate positioning of an additional aromatic side chains thereby enhancing the size of the aromatic cluster.

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ASSOCIATED CONTENT

* Supporting Information S

Assigned 1D 1H NMR spectra in chloroform and methanol, partial ROESY spectrum showing Cδ,δ′ H2 and Cβ H2 protons, F2 dimension cross sections of 2D HSQC-TOCSY, ROESY spectra in methanol, temperature, titration, aromatic proton chemical shifts, deuterium exchange plots, dihedral angles, and chemical shifts for peptide 2 and 2D HSQC-TOCSY for peptide 1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

Figure 10. Pathways of interconversion of conformer II to conformer I. Conformers (a) and (d) correspond to those observed in the two independent molecules in the crystallographicallic asymmetric unit of Boc-LFV-DPA-LFV-OME.31The two intermediate conformations were obtained by modeling, effecting a transformation from one crystallographically observed conformation to the other individually at Phe2 and Phe7. The corresponding side chain dihedral angles (°) for the conformations are χ1, χ2: (a) Phe2 = 178, 55 and Phe7 = −64, 91; (b) Phe2 = −65, 87 and Phe7 = −64, 91; (c) Phe2 = 178, 55 and Phe7 = −177, 69; and (d) Phe2 = −65, 87 and Phe7 = −177, 69.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank S. Aravinda for several discussions of hairpin crystal structures. R.S. acknowledges N. Chandrasekhar from Bruker, India for help in the NMR experiments. R.S. is supported by the award of a Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), India. This work is supported by a program in the area of Molecular Diversity and Design funded by the Department of Biotechnology, Government of India.

interconversion and alternatives in which dynamic motions of the phenyl rings are independent. It must be pointed out that phenylalanine side chain dynamics involves two distinct processes involving conformational interconversion between different χ1 states and the 180° flipping about the torsion angle χ2 (Cβ-Cγ). The χ2 flipping motion is extremely rapid on the NMR time scale resulting in chemical shift averaging yielding a single resonance for Cδ,δ′ H2 proton resonances of Phe7. The elegant work of Wüthrich and co-workers on BPTI and Szyperski and co-workers has provided estimates of the flipping rates for Phe rings in proteins.57,58,63 A flipping rate of approximately 102 to 105 per second even at −15 °C may be anticipated for an unhindered phenylalanine ring. The g−/t interconversion about the Cα-Cβ (χ1) bond is also likely to involve barriers which correspond to rapid exchange between conformational states on the NMR time scale. A particularly noteworthy feature of the temperature dependence of the ring current shifted proton CδH proton of Phe7 in both peptides is the downfield shift observed at temperatures below −33 °C. These observations suggest altered Phe ring orientation upon cooling. If a specific intermediate orientation is maintained as a consequence of an energetically favorable aromatic−aromatic interaction, the temperature dependence suggests a strong entropic contribution to the free energy. The observed chemical shift dependence suggests that the close approach of aromatic rings across the antiparallel strands is less favorable at low temperatures. A possible explanation that may be advanced is

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NMR Analysis of Cross Strand Aromatic Interactions in an 8 Residue

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