Trp–Trp Interactions in

Apr 7, 2015 - peptides,21 the stabilizing role of Trp−Trp pairs for β-hairpin structures has been ... interactions, Trp occupancy for packing in Fa...
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NMR Analysis of Tuning Cross-Strand Phe/Tyr/Trp−Trp Interactions in Designed β‑Hairpin Peptides: Terminal Switch from L to D Amino Acid as a Strategy for β‑Hairpin Capping Kamlesh M. Makwana and Radhakrishnan Mahalakshmi* Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal 462023, India S Supporting Information *

ABSTRACT: Interaction among the side chains of aromatic amino acids is a wellknown mechanism of protein and peptide structure stabilization, particularly in β sheets. Using short β-hairpin models bearing the sequence Ac-Leu-Xxx-Val-DProGly-Leu-Trp-Val-NH2, we report the surprising observation of significant destabilization in aryl−tryptophan interactions, which results in poorly folded peptide populations accompanied by lowering of stability. We find that such destabilization arises from forced occupancy of the indole ring in the shielded Edge position, in T-shaped aryl geometries. We demonstrate that this destabilizing effect can be efficiently salvaged by replacing the N-terminal LLeu with DLeu, which causes an increase in the folded hairpin population, while retaining Trp in the Edge position. Our observation of unique cross strand NOEs and data from temperaturedependent NMR and CD measurements reveals the formation of a locally stabilized aliphatic−aromatic network, leading to an overall increase in ΔGF° by ∼ −0.6 to −1.2 kcal/mol. Our results suggest that a contextual evaluation of stabilization by tryptophan is necessary in β hairpins. Furthermore, we report for the first time that the use of D isomers of aliphatic amino acids at the terminus is stabilizing, which can serve as a new strategy for increasing β-hairpin stability.

1. INTRODUCTION Rational design of synthetic peptides has emerged as a powerful approach in addressing the protein folding problem and in deducing the factors that govern protein folding and stability. These design strategies have set the rules for the construction of well-defined structural scaffolds, such as the use of strong two-residue turn nucleators for β-sheets with a DPro-Xxx (where Xxx = Gly, Pro, Aib, Ala), Aib-DAla, Asn-Gly, etc. sequence and helix nucleation and stabilization using Aib (and higher order analogues), Leu, Ala, and β and γ amino acids, to name a few.1−3 These selectively positioned residues direct the nature of the fold, whereas interactions among side chains (π···π, cation···π, S−H···π, C−H···π, etc.)4−7 and side chain substitutions (cyclization, halogenation, cross-linking, etc.) direct the stability.8−10 Structurally constrained β-hairpin scaffolds have served as excellent model systems to derive rules governing noncovalent interaction stereochemistry and free energy as well as for studying biomaterial interactions (DNA, RNA, metals, hydrogels, bioactive peptides, etc.).11−14 These scaffolds are usually derived from naturally occurring motifs in proteins, including BHKE, 15 B1 domain hairpin of protein G, 16−18 and ubiquitin.19,20 With the first report of de novo designed Trpzip peptides,21 the stabilizing role of Trp−Trp pairs for β-hairpin structures has been widely acknowledged in various aspects of studies ranging from peptide/protein design to simulations.22,23 Conversely, there also are a few studies that highlight its role in © 2015 American Chemical Society

structure destabilization and conferring stability at the expense of function as well as deliberations on why the Trp−Trp element is not evolutionarily frequent in proteins.24,25 Trp−Trp pairs and other synonymous hydrophobic aryl clusters engineered at the non-hydrogen bonding position of βhairpin arms (strands) display a T-shaped interaction geometry that is by and large independent of the solvent. In such interactions, Trp occupancy for packing in Face geometry is high; however, less stable parallel-displaced geometries are not uncommon.18,26 Currently, there is limited evidence for preferential indole conformations in miniature β-hairpin models, wherein a single aryl interaction can be specifically engineered while concurrently placing Trp at the less-favored Edge position. To date, the consequences of Trp at the Edge position in an isolated T-shaped aryl interaction is less understood. Short octapeptide β-hairpins (the simplest antiparallel strands connected by a tight two-residue turn) have proved to be attractive systems to investigate such isolated aromatic interactions. In the present study, using model DPro-Glynucleated β-hairpins (Table 1), we delineate the destabilizing effect of aryl−Trp interactions in which the indole moiety of tryptophan is placed forcefully at the Edge position. Received: January 19, 2015 Revised: April 4, 2015 Published: April 7, 2015 5376

DOI: 10.1021/acs.jpcb.5b00554 J. Phys. Chem. B 2015, 119, 5376−5385

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The Journal of Physical Chemistry B Table 1. Designed β-Hairpin Peptides

assignment and sequence-specific connectivity was achieved using a combination of 2D homonuclear 1H−1H TOCSY, 1 H−1H ROESY, and heteronuclear 1H−15N HSQC/1H−13C HSQC-TOCSY experiments. All 2D NMR spectra were recorded in phase-sensitive mode using TPPI methods. Solvent suppression was achieved using the standard excitation sculpting program available in the Bruker library. Mixing times of 80 and 250 ms were used for TOCSY and ROESY experiments, respectively. All NMR data processing was done with Topspin v3.0 software. 2.3. Structure Calculation. Solution NMR structure calculation was performed using CYANA v2.1 software.27 All NOEs observed in the ROESY spectrum were visually classified as strong, medium, and weak and assigned the upper distance limit as 2.5, 3.5, and 5.0 Å, respectively. The structures were further refined by adding hydrogen bonding and φ-angle constraints, the information for which was obtained from variable-temperature experiments used to monitor temperaturedependent amide chemical shifts and from the analysis of 3 JNH−CαH coupling constants, respectively. Calculations were performed until a zero violation state of distance and angle constraints was achieved. A total of 100 structures were calculated, and the best 35 structures were selected and superimposed using PyMOL.28 Average dihedral angle and RMSDs for the best 35 structure were calculated by MOLMOL.29 2.4. Chemical Shift Indexing and Free Energy Calculations. The deviation of observed CαH chemical shifts from random coil chemical shifts is one of the simplest methods for estimating secondary structure information and is known as chemical shift indexing (CSI).30,31 CSI calculations were carried out by comparing the observed CαH chemical shifts for the peptides with random coil chemical shifts obtained from the BMRB database (Biological Magnetic Resonance Bank)32 as well as from unfolded peptide controls synthesized using LP−G in the turns31,33 (Tables 1 and S1, Supporting Information). Furthermore, the observed CαH chemical shifts of residues 2, 3, and 6,34 relative to random coil and β-sheet chemical shifts obtained from the BMRB database,32 was used to calculate f F (fraction-folded population), Keq values (equilibrium constant), and ΔGF° (free energy of folding), using the following equations:7

a

Each hairpin is nucleated by the central DP−G turn sequence. Residues in bold blue represent the aryl residues at the non-hydrogen bonding position; residues in bold red represent the position in the hairpin, as shown in the schematic (right), where L↔D amino acid substitution was done. bAnalogs of peptides 1−6 with LP−G in the turn region were also made in order to represent the unfolded controls that lack a β-hairpin structure and cross-strand aromatic interactions. These peptides were labeled 1a−6a. See the Supporting Information for details.

Furthermore, in an endeavor to achieve hairpin stabilization, especially at the terminus, we purport to offset this destabilization by ingeniously engineering an aliphatic− aromatic microcluster by incorporating a terminal D amino acid.

2. MATERIALS AND METHODS 2.1. Peptide Synthesis and Purification. All peptides were synthesized on Rink amide AM resin (Novabiochem; EMD Merck Millipore) using standard solid-phase Fmoc chemistry.7 Activation of Fmoc-protected amino acids was carried out with HATU, HOBt, and DIPEA in DMF. Fmoc deprotection was achieved using 20% piperidine in DMF for ∼10 min and was constantly monitored with UV absorbance at 304 nm. Each amino acid coupling reaction was 45 min and was carried out twice. All peptides were acetylated at the N terminus with 5% acetic anhydride containing 3% DIPEA in DMF for 30 min. After completion of synthesis, the resin was incubated in a cleavage cocktail comprising 88:5:5:2 TFA/ phenol/water/TIPS for 3−4 h. TFA was evaporated in a rotatory evaporator, and the crude peptide was precipitated using cold diethyl ether. This was then purified by reversephase HPLC on a C18 column using methanol−water gradients (Figure S1, Supporting Information). Successful synthesis of all peptides was further confirmed by mass spectrometric analysis on an ion trap mass spectrometer (Esquire 3000+ ion trap, Bruker Daltonics, Figure S2, Supporting Information). Purified peptides were lyophilized to obtain white powders. Peptides 5− 7 were commercially synthesized and obtained in purified form. Mass spectra and HPLC profiles were obtained as described for peptides 1−4 (Figures S1−S2, Supporting Information). 2.2. NMR Spectroscopy. All peptides were structurally characterized by NMR spectroscopy. All 1D and 2D NMR experiments were recorded at 303 K on Avance III 500 and 700 MHz spectrometers (Bruker Biospin). NMR samples were prepared by dissolving lyophilized peptides to a concentration of ∼3 mM in CD3OH (99.8% D). Temperature-dependent proton chemical shifts were monitored by recording 1D spectra at temperatures ranging from 223 to 323 K in 10 K increments. All spectra were calibrated using TMS (0.0 ppm) or residual CH3OH resonance at 3.316 ppm. Complete resonance

fF = (δO − δ U)/(δ F − δ U)

(1)

Keq = fF − Av /(1 − fF − Av )

(2)

ΔG F° = −RT In Keq

(3)

Here, δO, δU, and δF are the observed, unfolded, and folded chemical shifts of a particular residue. R is the universal gas constant (1.987 cal/mol) and T = 303 K. f F values were averaged across positions 2, 3, and 6 to obtain f F−Av. These three positions were specifically chosen to represent the entire peptide, as they occupy key positions in the hairpin structure (residues 3 and 6 are in the hydrogen-bonding position) and have been accepted as most reliable in obtaining estimates of the folded population for such short sequences.34 2.5. Circular Dichroism Spectroscopy. Circular dichroism (CD) spectra were recorded on a JASCO J-815 spectropolarimeter equipped with a temperature-controlled Peltier setup. CD spectra were acquired using a 1 mm quartz cuvette between 195 and 260 nm at a scanning speed of 100 nm/min, a bandwidth of 1 nm, and a data pitch of 0.5 nm and 5377

DOI: 10.1021/acs.jpcb.5b00554 J. Phys. Chem. B 2015, 119, 5376−5385

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highly soluble and structured (Figure S3, Supporting Information). We achieved complete assignment of all resonances using a combination of TOCSY, ROESY, and HSQC experiments (Figures S4−S6, Supporting Information). Information on hydrogen-bonded versus solvent-exposed amides was obtained using temperature-dependent 1D measurements (Figure S7, Supporting Information). The overall summary of the 2D NMR data, marking the structurally relevant β-hairpin NOEs, is represented schematically in Figure 2 (also see Figure S5, Supporting Information). Interstrand NOEs between residues 3−6, 2−7, and 1−8 as well as the uncommon 2α−8NH NOE (green arrows in Figure 2) clearly indicate the formation of a β-hairpin structure for peptides 1, 3, and 5. However, apart from the 1NH−2NH NOE, we also observe the 6NH−7NH NOE (highlighted as an orange arrow in Figure 2) in the case of peptides 1 and 3, which suggests the possible occurrence of strand fraying in these peptides. Overall, our NMR data indicate that DP−G-nucleated βhairpin structures are formed in all peptides (Figure S8, Supporting Information). Comparison of fraction-folded and free energy values derived from NMR chemical shifts reveals an increase of ∼ −0.4 and ∼ −0.7 kcal/mol for peptides with Phe−Trp (peptide 3) and Trp−Trp (peptide 5) interactions, respectively, over the Tyr−Trp (peptide 1) pair (Figure 4, section 3.3). However, when we compare these values with reported stabilizing contributions from Trp−Phe37 or Trp− Tyr38 pairs, we observe a difference of ∼2.0 kcal/mol. This suggests that aryl−Trp interaction in our peptides is not stabilizing; indeed, it results in a lower population of folded peptides as compared to previous reports. Hence, we investigated the source of this destabilization by examining the behavior of aryl groups using NMR. 3.2. Nature of Cross-Strand Aromatic Interactions in Peptides 1, 3, and 5. In FtE T-shape aromatic interactions, studies from our group as well as others have reported that the Edge aryl resonances undergo upfield shifts because of the proximity of the aryl Face π cloud.4,14,34,39−43 Indeed, examination of the 1D 1H NMR spectra of the three peptides reveals that 7Cε3H is upfield-shifted (Figure S3, Supporting Information). Furthermore, the presence of characteristic NOEs 2Cδ/ε/ε3H−7CαH and 2CδH−7Cε3H confirms the existence of T-shape aromatic interactions (Figure S5, Supporting Information). The 2Cδ/ε/ε3H−3NH/5CαH/6NH NOEs and NOEs between Trp7 aryl resonances to 1NH and the N-terminal acetyl group position rings 2 and 7 near the turn and termini, respectively (Figure S5, Supporting Information). Most surprisingly, investigation of the chemical shift pattern provides us with an intriguing upfield-shifted aryl 2 resonance in all three peptides (1, 3, and 5), which is in addition to the anticipated upfield-shifted W7 resonances (Table 2). Aryl 2 Cα/β/δ/ε3H is upfield-shifted in the order 1 > 3 > 5, and the marginal upfield shift of W7 Cα/β/ε3H follows the order 5 > 3 > 1 for the three peptides. Moreover, the upfield shift for W7 Cα/β H, expected in FtE interactions, is prominent and temperature-dependent only in 5 (Figure S9a−c, Supporting Information). Our NMR data therefore provides us with alternate orientations of the two aryl rings, which gives rise to two peptide populations in solution. The major population possesses T-shaped aryl interactions with W7 as the Edge. However, a minor population exists, wherein W7 adopts either a Face occupancy or a parallel-displaced π···π interaction. In both the latter cases, shielding of 2Cα/β/δ/ε3H by the W7 π

were averaged over three acquisitions. Samples were prepared by dissolving lyophilized peptides in methanol. Although all the peptides were highly soluble in methanol, we encountered difficulties when dissolving the samples in water because of the highly apolar nature of the designed sequences. We therefore used 0.1% TFA in water in order to sufficiently solubilize the peptides and obtain concentrations adequate for CD measurements. Peptide concentrations were determined spectrophotometrically using sample absorbance measured at 280 nm. Temperature-dependent CD measurements were carried out using temperature-controlled interval scan measurements between 195 and 260 nm at temperatures ranging 5−71 °C with a ramp rate of 2 °C/min in methanol and 5−95 °C with a ramp rate of 5 °C/min in water/TFA. Data acquired at each temperature point were averaged over three acquisitions. Blanksubtracted, smoothed spectra were then converted to molar ellipticity values using reported methods.7 All plots were generated using SigmaPlot v10.0 (SYSTAT Software).

3. RESULTS AND DISCUSSION 3.1. Backbone Conformation of Peptides 1, 3, and 5. The interaction among aromatic side chains, particularly Trp, placed at the non-hydrogen-bonding position in β hairpins plays an important role in structure stabilization and generally displays a T-shaped geometry (Figure 1).14 Being bulky in size

Figure 1. T-shaped aromatic interactions. T-shape interaction in peptide hairpins can be further distinguished as Face-to-Edge (FtE) or Edge-to-Face (EtF) interactions. Interaction is FtE when the Face aromatic is at the N-terminal strand and EtF when the Edge aromatic is at the N-terminal strand. Shown here are schematic representations of FtE and EtF aromatic interactions (left and center) and a typical FtE aromatic interaction in hairpins wherein the second residue (Tyr) at the non-hydrogen bonding position (NHB) in the N-terminal strand takes up Face geometry, and the seventh residue (Trp) at NHB in the C-terminal strand takes up Edge geometry.

and amphipathic in nature, tryptophan displays a multitude of interactions with its immediate surroundings. In such interactions, it has been experimentally observed that Trp prefers packing in the Face geometry,26,35 wherein the indole also contributes sizably to the driving of folding and stability of short peptide scaffolds.36 However, what happens when a Trp ring is forcibly placed in Edge position in a defined structural βhairpin scaffold is less understood. We therefore designed peptides 1, 3, and 5 to systematically investigate the mode and impact of Face-to-Edge (FtE) Tyr−Trp, Phe−Trp, and Trp− Trp interactions, respectively, with Trp in Edge geometry in short β-hairpins (Table 1). We observed sharp, well-resolved resonances in the NMR spectra for the peptides, suggesting that the three sequences are 5378

DOI: 10.1021/acs.jpcb.5b00554 J. Phys. Chem. B 2015, 119, 5376−5385

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Figure 2. Schematic representation of structurally relevant NOEs observed in the homonuclear 1H−1H ROESY spectrum of peptides 1 (left), 3 (middle), and 5 (right). Green arrows, diagnostic β-hairpin NOEs; blue arrows, aromatic-to-backbone NOEs; red arrows, aromatic−aromatic NOEs; and orange arrows, NOEs indicating possible strand fraying.

Table 2. Comparison of 1H NMR Chemical Shifts across the Peptides at Positions 2 and 7 with Respect to Random Coil Values protonsa 2 7 2 7 2 7

CδH/Cε3H Cε3H CβH CβH CαH CαH

1

2

RCb

3

4

RCb

5

6

7

RCb

6.51 7.27 2.72/2.22 3.10/3.01 4.72 4.95

6.81 7.32 2.93/2.53 3.20/3.04 5.09 4.76

7.02 7.65 2.91/2.64 3.16/2.93 4.46 4.58

6.83 7.18 2.92/2.41 3.12/2.96 4.99 4.84

7.04 7.24 3.67/2.67 3.20/2.99 5.22 4.72

7.26 7.65 3.03/2.75 3.16/2.93 4.56 4.58

7.42 6.77 3.29/2.73 2.87/2.43 5.02 4.70

7.54 6.78 3.40/2.90 2.90/2.37 5.17 4.49

7.53 7.02 3.38/2.92 2.92/2.40 5.19 4.48

7.65 7.65 3.16/2.93 3.16/2.93 4.58 4.58

a

All values are for data recorded at 303 K. bRandom coil (RC) values.44 A complete list of standard chemical shifts for folded and RC structures is available at the BMRB. Anomalous upfield-shifted protons with respect to random coil values indicate mode and extent of aromatic interaction in individual peptides. Values highlighted in bold, red in peptides 1, 3, and 5 represent unexpected chemical shifts for peptide β hairpins with a typical FtE aryl geometry.

Figure 3. CαH chemical shift indexing. Residue-wise variation of CαH chemical shifts with respect to random coil chemical shifts are compared across different peptides. (a) Random coil values for the respective residues obtained from the BMRB database were used to generate the CSI plots. (b) Chemical shift values from the respective unfolded (LPro−Gly) analogues (provided in Supporting Information) were used for the calculations. A similar pattern observed in both calculations indicates that random coil values derived from protein structures could reliably be employed to derive meaningful CSI information for these peptides in methanol. Greater positive values of 2 vs 1, 4 vs 3, and 6 vs 5 indicate that the DLeu analog of peptides are well-folded and more stable as compared to the LLeu counterparts. In addition, it is noteworthy that the preceding D residue does not significantly influence the succeeding Hα chemical shifts. The substantial difference in CSI values at R2 seen in (a) between 1 vs 2, 3 vs 4, and 5 vs 6 are retained in calculations using the respective control peptides (b).

cloud can give rise to our observed anomaly in the aryl 2 shifts. We map the differences in aryl interaction strengths to arise

from a competition of tryptophan for the Face geometry, which causes shielding of the aryl 2 resonances in the order Tyr > Phe 5379

DOI: 10.1021/acs.jpcb.5b00554 J. Phys. Chem. B 2015, 119, 5376−5385

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The Journal of Physical Chemistry B > Trp. A consequence of this χ1 conformational equilibrium (FtE and Edge-to-Face (EtF) ring orientation interconversion arises from concurrent rotation at χ1)35,45 is the lowering of ΔGF° for the three peptides (described in section 3.1). Similar observations have been made in other peptide hairpin systems.18,35,45 3.3. Engineering N-terminal DLeu1 Peptide Analogs. Tremendous research on understanding and engineering the factors that stabilize β hairpins have led to promising results in this field.14 Short peptide hairpins face the common problem of strand fraying at the termini. Particularly, in our peptides 1, 3, and 5, because of the additional aryl χ1 conformational dynamics, terminal destabilization and peptide unfolding is pronounced at higher temperatures. (See dδ/dT values in Figures S3 and S7, Supporting Information.) In an attempt to increase strand stabilization and minimize ring dynamics, we introduced an aliphatic−aromatic cluster by reverse-engineering the chiral center at the N-terminal residue. The sequences of peptides 2, 4, 6, and 7, designed on the basis of the parent peptides 1, 3, and 5, are provided in Table 1. The extent of folding in each peptide can be qualitatively indexed by comparing the deviation of CαH chemical shifts from random coil values through CSI. Proton chemical shifts are known to be influenced by various factors including the solvent system.46 To rule out possible solvent contributions (BMRB data set values are primarily obtained from aqueous media) or the influence of a proximal D residue on the CSI, we used two sets of random coil values: one was derived from the BMRB database and the other was obtained from control peptides that were unstructured (listed in Table S1, Supporting Information). CSI comparison shown in Figure 3 (and Figure S10, Supporting Information) clearly suggests that peptides 2, 4, and 6 (DLeu1 analogues of peptides 1, 3, and 5, respectively), with a greater overall positive value in CSI, form well-structured β hairpins more stable than those from its LLeu1 analogs. Further confirmation is obtained from the number and strength of long-range NOEs obtained in these peptides (Figure S5, Supporting Information). Finally, NMR structural evidence indicates an increase in the calculated stability of peptides 2, 4, and 6 by ∼0.6 kcal/mol for the Tyr/Phe−Trp pair and ∼1.0 kcal/mol for the Trp−Trp pair over peptides 1, 3, and 5, respectively (Figure 4). An additional ∼0.2 kcal/mol increase is seen in peptide 7 (over peptide 6), in which case the DLeu1 is replaced by a DIle1. On the basis of the calculated ΔG°F , we obtain a rank order for peptide stability: 7 > 6 > 4 > 5 > 2 > 3 > 1. 3.4. Formation of an N-Terminal Aliphatic-Aromatic Microcluster. The extent of upfield shift of an aromatic resonance involved in T-shaped interactions has been shown to correlate with the geometry and strength of aromatic interaction.31 We anticipated that peptides with greater ΔGF° would also display stronger aromatic interactions and more upfield-shifted resonances. Interestingly, however, when we compare the aromatic proton chemical shifts for W7 Cε3H across all peptides (Table 2), the magnitude of upfield shift for this resonance is not significantly different at 303 K in the LLeu and DLeu analogs. Furthermore, although the W7 CβH is marginally downfield-shifted in the D analogs when compared with the L analogs, the W7 CαH shows an upfield shift (Table 2). How do the DL/DI1-containing sequences therefore display greater ΔGF° values? To address this, we further examined the NMR spectra of these peptides. Surprisingly, these constructs

Figure 4. Comparison of fraction-folded, free energy values and the extent of upfield-shifted signature aromatic proton resonances. With an increase in the folded fraction (upper panel, squares connected with dashed lines), the folding free energy value shows a concomitant and favorable increase, as seen from the more negative ΔG°F from peptide 1 to 7 (upper panel, circles connected with red solid line). This correlates well with the chemical shift deviation of the aryl ring protons of second and seventh residues (lower panel; values for second residue, circles, and those for seventh residue, squares). W7 Cε3H shows a larger deviation from random coil values in peptides 5−7, suggesting that in a large population of the molecules, W7 ring adopts the Edge position, contributing to peptide stabilization.

do not display anomalous behavior of aryl 2 resonances (Table 2); for instance, temperature dependence of Y/F2 CδH and W7 Cε3H of 1 versus 2 and 3 versus 4 highlights this difference (Figures 5 and S11a,b, Supporting Information). Hence, a part of the stabilization could arise from considerably abolishing the FtE↔EtF interconversion of the aryl rings seen in peptides 1, 3, and 5. Furthermore, the side chain resonances of DL/DI1 are also upfield-shifted as compared to those of LL (Figure S3, Supporting Information). Such behavior of aliphatic resonances is also a good indicator of ring current effects, and may relate to the occurrence of local C−H···π interactions. Figure 6 illustrates the number of observed NOEs across peptides 5−7. The side chains of DL/DI1 show considerable occupancy in the proximity of the indole rings (Figure 6), which could result from the formation of stable C−H···π interactions. Similar interactions are known to stabilize cis Pro− Trp bonds in short peptides.47 On the basis of the information from observed NOEs between DL/DI1↔W7, upfield shifts of W7 ring protons and the conspicuous reduction in the anomalous shifts in aryl 2, we confirm the formation of a stabilizing C−H···π···π network, in peptides 6 and 7 (Figure 6). Furthermore, increase in the overall scaffold stability clearly suggests that the formation of the C−H···π···π network is a greater contributor to β-hairpin strength. Similar NOEs are also observed in peptides 2 and 4 (Figure S5, Supporting Information). The observed ΔGF° rank order (7 > 6 > 4 > 2) is explained by the additive effect of several weak C−H···π interactions. 3.5. Side Chain Orientation of Trp−Trp Pair in 5−7. We further analyzed specific preferences in ring geometry and Trp−Trp interaction in peptides 5−7 (Figures 7 and S11, 5380

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Figure 6. Observation of strong aliphatic ↔ aromatic NOEs in peptides (a) 5, (b) 6, and (c) 7 in the homonuclear 1H−1H ROESY spectra of the three peptides acquired at 303 K. All NOE intensities were adjusted to the 3α ↔ 4δ NOE intensity, which is shown separately on the left. The number and intensity of NOEs obtained for each peptide among residue 1 and the aryl groups of W2 and W7 increases between peptide 5 and 6, which supports the formation of a local C−H···π microcluster in peptide 6. Also, note the marginal upfield shift of the DL1 resonance, resulting from the proximity to the π cloud of the aryl rings. Substitution of DL1 with DI1 further strengthens local C−H···π interactions, observed by the additional demarcation of methyl resonances in peptide 7.

Figure 5. Plot of temperature-dependent chemical shift variation of the signature upfield-shifted aromatic protons. Apart from upfieldshifted W7 Cε3H (which is the signature for T-shape FtE geometry in our peptides), the temperature-dependent upfield shifts of 2δH also highlight the existence of reverse EtF geometry in peptides 1−4. Note that in DLeu analogs (peptides 2 and 4) aryl 2 CδH is not only less temperature-dependent but is also downfield-shifted as compared to its LLeu peptides (1 and 3, respectively) suggesting that the population with reverse EtF geometry is considerably reduced.

Supporting Information), using the anomalous shifts of W2 and W7 Cε3H (the resonance that is most affected in T-shaped aromatic interactions).34,39−42 At 303 K, the 7Cε3H is upfieldshifted by ∼ −0.88, ∼ −0.87, and ∼ −0.63 ppm for 5−7, respectively, with respect to the random coil chemical shift of 7.65 ppm. In comparison, the 2Cε3H displays a marginal shift of ∼ −0.23, ∼ −0.11, and ∼ −0.12 ppm for 5−7, respectively. Hence, in all three peptides, the predominant population displays Face occupancy for W2, and the π cloud of this indole shields W7 Cε3H (W7 Edge). Most interestingly, however, the extent of 7Cε3H upfield shift is highest for peptide 5; furthermore, this resonance undergoes a considerable anomalous upfield shift upon lowering the temperature only in this peptide (dδ/dT for this resonance across the three peptides are 11.2, 9.0, and 5.4 ppb/K for 5−7, respectively). Such substantial temperature dependence for peptide 5 (as against 6 and 7) suggests that the population of peptides displaying T-shaped Trp↔Trp interaction as well as the strength of this interaction augments upon cooling. Hence, the χ1 motion of W2 and W7 is lowered, with the population now adopting the stable FtE geometry. Peptides with the D residue at the N terminus have lowered W2π···W7Cε3H interaction, which may be offset by a minor stable population displaying 2π···7Cδ1H interaction in the order 7 > 6 > 5. We reach this conclusion on the basis of upfield shift observed for the W7 Cδ1H resonance and the reduction in exciton couplet at 227 nm in our experiments (discussed in section 3.6). Hence, we do not rule out the possibility that peptides 6 and 7 possess a ring-flipping motion around χ2 of W7, which is sterically restricted by the proximity of the DL/DI1

side chain. Availability of (alternate) C−H···π interaction for W7 could result in lowered π···π interaction strength for peptides 6 and 7, particularly at low temperatures. 3.6. Monitoring Aromatic Interactions using CD Thermal Melting. Previous work from our laboratory and others has established the overwhelming contribution of interacting aryl pairs to the far-UV region of the peptide electronic CD spectra.38,40,48 Such Cotton effects arise from nondegenerate exciton interactions between two chromophores in a chiral local environment, which in this case is presented by the peptide backbone.49 As the peptide unfolds, a loss in the CD signal is anticipated. Particularly, Trp−Trp pairs display a signature exciton couplet with negative and positive maxima at 214 and 227 nm, respectively.41,48 In line with previous studies, we observe a considerable contribution of aryl interactions in the form of a bisignate exciton couplet in the far-UV CD spectra for all the peptides (Figure 8). Interestingly, the CD spectra recorded in both methanol and water (+0.1% TFA) are similar, indicating that aromatic ring orientations are comparable in both solvents and suggesting that the peptides retain structure even in aqueous solvents. It must be noted here that the design and study of very short peptide β hairpins that fold well in water or aqueous media is in itself a challenge. In methanol, comparison of the molar ellipticity (ME) values suggest that LLeu analogs of peptides 1, 3, and 5 display stronger aromatic interactions as compared to those of the DLeu analogs (2, 4, and 6). 5381

DOI: 10.1021/acs.jpcb.5b00554 J. Phys. Chem. B 2015, 119, 5376−5385

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Figure 7. Temperature dependence of aromatic proton chemical shifts. Stack plots of the 1H 1D spectra of peptides (a) 5, (b) 6, and (c) 7, highlighting the variation of aryl proton resonances with temperature. Resonances that show substantial temperature dependence are marked in red, and the movement of the resonance is traced using red dotted lines. (d) Comparison of the Cε3H resonance of residues 2 and 7 across peptides 5−7. Note that 2Cε3H is largely invariant in the three peptides across a >100 K change in temperature. However, the 7Cε3H chemical shift shows a remarkable upfield shift upon lowering of temperature, caused by the shielding effect of the W2 ring. Of the three peptides, the largest upfield shift is observed for peptide 5, followed by those of 6 and 7; the extent of this anomalous shift is a known indicator of strong T-shaped aromatic interactions. (See text for details.)

to the CD spectrum is highest for peptide 5, the midpoint of thermal denaturation (Tm) is surprisingly the lowest (Tm = 46, 57.8, and 56.05 °C for 5−7, respectively). The low Tm is in good agreement with NMR analysis of secondary structure and overall peptide stability (discussed earlier). The contrast in increased CD contribution versus the secondary structure analysis and overall peptide stability, which is lowest for 5 among the Trp−Trp peptides (5−7), merits attention. It is evident that our CD measurements monitor the aromatic contribution to the far-UV spectrum and not necessarily the overall scaffold stability. The CD thermalunfolding profiles, combined with the extent of anomalous shifts for the aryl 7 resonance (Figures 7 and S11, Supporting Information), suggest that aromatic interactions are strongest in peptide 5. However, this indole interaction occurs at the expense of a stable β-hairpin scaffold. (See ΔGF° in Figure 4 and Tm values.) It is likely that in peptide 5, aryl interactions persist despite considerable peptide unfolding, giving rise to residual CD signal even at 95 °C (Figure 9). Weaker ellipticity in peptides 6 and 7 may arise from the presence of alternate W7 ring geometry obtained by ring-flip at χ2, which is speculated to abolish the exciton contribution.35 Nevertheless, weaker aromatic interactions in 6 and 7 are compensated for by C− H···π interactions and result in the observed increase in folded

Figure 8. Far-UV CD spectra of the various peptides recorded in methanol (left) and water (containing 0.1% TFA) (right). Peptides with strong Trp−Trp interactions, when placed in an achiral environment, show considerable exciton contribution to the CD spectrum, with maxima at 214 and 227 nm. Note how the aryl interactions are largely conserved across water and methanol in peptides 5−7.

Owing to the presence of two indole moieties, peptides 5−7 show a two- to threefold increase in the exciton contribution to CD. Furthermore, upon thermal denaturation in both methanol and water (+0.1% TFA), we obtain a sigmoidal temperaturedependence of the 227 nm band, indicating that the peptides undergo a simple two-state unfolding (Figures 9 and S12, Supporting Information). Although the aromatic contribution 5382

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Figure 9. Thermal denaturation studies for peptide 5 (left), 6 (middle) and 7 (right) recorded in water (containing 0.1% TFA). Top panels show the wavelength scans highlighting the loss of exciton contribution as the temperature is varied from 5 °C (purple) to 95 °C (red) by increments of 5 °C. Note how the initial molar ellipticity (ME) values are highest for peptide 5 and is completely abolished at 95 °C only for peptide 6. Lower panels show the change in ME for the three peptides at 227 nm (red symbols) fitted to a sigmoidal function (black line) to derive the midpoint of thermal denaturation in each case.

β-hairpin populations through additional backbone-stabilized interactions. (See ΔG°F in Figure 4; see also Figure 6.)

likely to be the reason why tryptophan, when observed in proteins, preferentially displays a Face occupancy.50 It has been suggested that the FtE ↔ EtF interconversion requires unfolding of the backbone.35 In proteins, it is an intriguing possibility that deliberate positioning of Trp residues at the Edge be used to introduce local perturbations and provide the conformational dynamicity necessary for function. Particularly, the documented loss-of-function upon introduction of cross-strand Trp−Trp interactions, such as that observed in the hPin1 WW domain,25 may be offset by Phe/ Tyr−Trp interactions. Conformational interconversion at χ1 of the aryl rings lowers the otherwise favorable contributions of aromatic interactions at the non-hydrogen-bonding position. Such FtE ↔ EtF flipflop interconversion also involving a turned rotamer for tryptophan at χ2 has been observed earlier.35 We also observe alternate χ2 for W7 in a minor population of peptide 7, which is evident from the upfield shift of the 7Cδ1H signal (Figure 7); such geometries are also possible in proteins (Figure 11). We have also established through this study that peptide systems containing less favorable aryl−Trp interactions can be readily rescued by providing the indole with alternative interaction elements, achieved by the mirroring of a single residue, without altering the sequence length. The resulting aliphatic−aromatic microcluster (Figure 10b) can also be used as a strategy in proteins to impart local stabilization to the folded protein core through establishment of multiple weak contacts, similar to β-capping motifs52,53 and hydrophobic clusters54 designed earlier. It is of interest to note that multiple weak C−H···π interactions can together surpass the otherwise overwhelming stabilization conferred by electrostatic aryl interactions. Although C−H···π contacts are considered weak interactions, our study illustrates that this stabilizing factor although small in magnitude can possess an additive contribution to local interactions, and thereby contribute to the overall stability of

4. CONCLUSIONS We have demonstrated that the presence of tryptophan at the edge position of FtE aromatic interactions may impose alternate aryl interaction geometries (illustrated in Figure 10a) and lead to destabilization of short β hairpins. Hence, the influence of Trp residues on the overall β-hairpin stabilization is contextual. The disposition for alternate geometries is also

Figure 10. (a) Modeled structures of peptide 1 showing interconversion of geometry from FtE (salmon) to EtF (green) interaction as a representative of what seen in Aro−Trp pairs. Arrowheads represent the coordinated side-chain interconversion during the FtE ↔ EtF flip-flop. (b) Representative distances in calculated NMR structures for peptides 5 (magenta), 6 (green), and 7 (cream), highlighting the formation of a C−H···π···π microcluster when DL is the first amino acid. 5383

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(4) Tatko, C. D.; Waters, M. L. Selective Aromatic Interactions in Beta-Hairpin Peptides. J. Am. Chem. Soc. 2002, 124, 9372−9373. (5) Tatko, C. D.; Waters, M. L. Investigation of the Nature of the Methionine-Pi Interaction in Beta-Hairpin Peptide Model Systems. Protein Sci. 2004, 13, 2515−2522. (6) Riemen, A. J.; Waters, M. L. Design of Highly Stabilized BetaHairpin Peptides through Cation-Pi Interactions of Lysine and NMethyllysine with an Aromatic Pocket. Biochemistry 2009, 48, 1525− 1531. (7) Makwana, K. M.; Raghothama, S.; Mahalakshmi, R. Stabilizing Effect of Electrostatic Vs. Aromatic Interactions in Diproline Nucleated Peptide Beta-Hairpins. Phys. Chem. Chem. Phys. 2013, 15, 15321−15324. (8) Osapay, K.; Tran, D.; Ladokhin, A. S.; White, S. H.; Henschen, A. H.; Selsted, M. E. Formation and Characterization of a Single Trp-Trp Cross-Link in Indolicidin That Confers Protease Stability without Altering Antimicrobial Activity. J. Biol. Chem. 2000, 275, 12017− 12022. (9) Cooper, W. J.; Waters, M. L. Turn Residues in Beta-Hairpin Peptides as Points for Covalent Modification. Org. Lett. 2005, 7, 3825−3828. (10) Santiveri, C. M.; Leon, E.; Rico, M.; Jimenez, M. A. ContextDependence of the Contribution of Disulfide Bonds to Beta-Hairpin Stability. Chemistry 2008, 14, 488−499. (11) Pantoja-Uceda, D.; Santiveri, C. M.; Jimenez, M. A. De Novo Design of Monomeric Beta-Hairpin and Beta-Sheet Peptides. Methods Mol. Biol. 2006, 340, 27−51. (12) Nagarkar, R. P.; Hule, R. A.; Pochan, D. J.; Schneider, J. P. De Novo Design of Strand-Swapped Beta-Hairpin Hydrogels. J. Am. Chem. Soc. 2008, 130, 4466−4474. (13) Robinson, J. A. Beta-Hairpin Peptidomimetics: Design, Structures and Biological Activities. Acc. Chem. Res. 2008, 41, 1278− 1288. (14) Santiveri, C. M.; Jimenez, M. A. Tryptophan Residues: Scarce in Proteins but Strong Stabilizers of Beta-Hairpin Peptides. Biopolymers 2010, 94, 779−790. (15) Pastor, M. T.; Lopez de la Paz, M.; Lacroix, E.; Serrano, L.; Perez-Paya, E. Combinatorial Approaches: A New Tool to Search for Highly Structured Beta-Hairpin Peptides. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 614−619. (16) Munoz, V.; Thompson, P. A.; Hofrichter, J.; Eaton, W. A. Folding Dynamics and Mechanism of Beta-Hairpin Formation. Nature 1997, 390, 196−199. (17) Olsen, K. A.; Fesinmeyer, R. M.; Stewart, J. M.; Andersen, N. H. Hairpin Folding Rates Reflect Mutations within and Remote from the Turn Region. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 15483−15487. (18) Andersen, N. H.; Olsen, K. A.; Fesinmeyer, R. M.; Tan, X.; Hudson, F. M.; Eidenschink, L. A.; Farazi, S. R. Minimization and Optimization of Designed Beta-Hairpin Folds. J. Am. Chem. Soc. 2006, 128, 6101−6110. (19) Platt, G. W.; Simpson, S. A.; Layfield, R.; Searle, M. S. Stability and Folding Kinetics of a Ubiquitin Mutant with a Strong Propensity for Nonnative Beta-Hairpin Conformation in the Unfolded State. Biochemistry 2003, 42, 13762−13771. (20) Riemen, A. J.; Waters, M. L. Stabilization of the N-Terminal Beta-Hairpin of Ubiquitin by a Terminal Hydrophobic Cluster. Biopolymers 2008, 90, 394−398. (21) Cochran, A. G.; Tong, R. T.; Starovasnik, M. A.; Park, E. J.; McDowell, R. S.; Theaker, J. E.; Skelton, N. J. A Minimal Peptide Scaffold for Beta-Turn Display: Optimizing a Strand Position in Disulfide-Cyclized Beta-Hairpins. J. Am. Chem. Soc. 2001, 123, 625− 632. (22) Mirassou, Y.; Santiveri, C. M.; Perez de Vega, M. J.; GonzalezMuniz, R.; Jimenez, M. A. Disulfide Bonds Versus Trp···Trp Pairs in Irregular β-Hairpins: NMR Structure of Vammin Loop 3-Derived Peptides as a Case Study. ChemBioChem 2009, 10, 902−910. (23) Koukos, P. I.; Glykos, N. M. Folding Molecular Dynamics Simulations Accurately Predict the Effect of Mutations on the Stability

Figure 11. Representative example of cross-strand Trp−Trp interaction between the π cloud of one indole and a Cδ1H of the second indole, as seen in the non-hydrogen bonding position of an antiparallel strand in a protein crystal structure (Protein Data Bank ID: 3MRK).51 Such indole interactions are less common compared to the interactions involving Trp Cε3H.

the scaffold. The use of branched-chain D aliphatics as capping residues can result in better terminal packing of even short octapeptides while also providing proteolytic stability,55,56 encouraging the rational design of stable peptide scaffolds.

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ASSOCIATED CONTENT

S Supporting Information *

Peptide synthesis, purification, and characterization, 1D and 2D NMR measurements, thermal denaturation studies from NMR, structure calculations and relevant NOE information, and CD studies. 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]. Funding

This work is supported by intramural funds. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.M.M. thanks the University Grants Commission, Government of India, for a senior research fellowship. R.M. is a recipient of a Ramalingaswami Fellowship from the Department of Biotechnology, Government of India.

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REFERENCES

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