Effects of the Terminal Aromatic Residues on Polyproline

Article pubs.acs.org/JPCB

Effects of the Terminal Aromatic Residues on Polyproline Conformation: Thermodynamic and Kinetic Studies Yu-Ju Lin,† Li-Kang Chu,†,‡ and Jia-Cherng Horng*,†,‡ †

Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 30013, R.O.C. Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan 30013, R.O.C.

‡

S Supporting Information *

ABSTRACT: In a peptide or protein, the sequence of aromatic residue−proline or proline−aromatic residue shows a high propensity in forming cis prolyl bonds due to aromatic−proline interactions. In this work, we designed and prepared the polyproline peptides with aromatic amino acids (F, Y, W) incorporated into their N-terminal or C-terminal end to investigate the effects of a terminal aromatic residue on polyproline conformation and the transition kinetics of polyproline I (PPI) to polyproline II (PPII) helices. Circular dichroism measurements reveal that the N-terminal aromatic−proline interaction imposes a more pronounced consequence on the forming propensity of PPI conformation than does the C-terminal aromatic−proline interaction in n-propanol. The propensity of forming PPI is correlated with the strength of aromatic−proline interactions in the order of Tyr-Pro > Trp-Pro > Phe-Pro. In aqueous solution, kinetic studies indicate that aromatic-substitution effects are nondirectional and indistinct on the PPI → PPII conversion rates, suggesting that aromatic−proline interactions may not be an important factor in this process. In addition, the temperature-dependent kinetics shows that the hydrophobicity of aromatic side chain may play a critical role affecting the activation enthalpy and entropy of the conversion of PPI to PPII, providing new insights into the folding of polyproline helices.

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INTRODUCTION Among the naturally coded amino acids, proline has a lower energy barrier than any other amino acids on the isomerization of a peptide bond due to a comparable steric conflict between cis and trans conformations.1 Thus, proline residue has the highest propensity to form a cis amide bond among the natural amino acids.2,3 In the model peptides and proteins reported in protein data bank, a high ratio of cis prolyl bonds was found in the proline−aromatic or aromatic−proline sequences, suggesting that favorable interactions between aromatic residue and proline could exist to enhance the population of cis peptide bonds.4 Such interactions are called aromatic−proline interactions that occur between the aromatic ring and proline, which are frequently found as a major pattern in protein−protein interactions and have an important contribution to protein folding stability.4−13 As shown in Figure 1, aromatic−proline interactions are largely from CH···π effects, in which the partially positive charged hydrogen(s) of a polarized C−H bond in proline interact with the π face of an aromatic ring.4,7,8,14,15 Meanwhile, the hydrophobic effects between the pyrrolidine ring of proline and aromatic residues also contribute to aromatic−proline interactions.4 Aromatic−proline interactions are in a similar manner comparable to cation−π interactions because that Hα and Hδ of proline are adjacent to the electron-withdrawing © XXXX American Chemical Society

Figure 1. Illustration of aromatic−proline interactions: Aromatic-cisPro configuration and cis- Pro-Aromatic configuration.

groups, carbonyl and amide nitrogen, making them most partially positive and interact favorably with aromatic rings.4 Aromatic− proline interactions could be also stabilized by the additional electron delocalization between the aromatic π orbital and the σ orbital of the C−H bond,7,8,14 and thus aromatic−proline interactions can be considered electrostatic (δ−aromatic − δ+proline) and partly stereoelectronic (πaromatic → σproline C−H) in nature.8 Studies of the SXPYDV peptide by Wright et al.,16 the AXPAK Received: September 7, 2015 Revised: December 6, 2015

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DOI: 10.1021/acs.jpcb.5b08717 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B peptide by Fischer et al.,17 and the GXPG peptides by Raleigh et al.18 showed a similar result that Trp-Pro and Tyr-Pro have a higher prolyl cis amide bond propensity than Phe-Pro, strongly suggesting that the strength of aromatic−proline interactions is related to aromatic electronics. Zondlo and co-workers have modulated aromatic−proline interactions by tuning the electron density of aromatic system with different substituents on the aromatic ring. Their results showed that an electron-rich aromatic ring could form stronger aromatic−proline interactions with a proline residue, while a weaker interaction was observed for an electron-deficient aromatic ring.4,15 Prolyl isomerization plays an important role in protein folding19−23 and is critical to the biological responses related to isomer-specific recognition.24−28 In proteins, proline-rich sequences often form polyproline II (PPII) structure, a lefthanded helix with all trans peptide bonds, as a recognition motif in the protein−protein interaction interface.28−30 PPII-type helices are also regarded as the predominated structures in unfolded proteins.22,31−34 In addition, oligoproline peptides have been widely used as rulers and scaffolds in various applications due to their conformational stability and rigidity,35−50 showing that oligoprolines are an attractive and valuable target for study. Furthermore, a crystal structure of an oligoproline PPII-helix was just reported,51 revealing useful structural information that would be taken into account for designing new PPII-based scaffolds. In contrast to PPII, polyproline I (PPI) structure that polyproline can also form is a right-handed helix with all cis peptide bonds and is less common. PPI and PPII adopt similar backbone dihedral angles, (ϕ, ψ) = (−75°, 160°) for PPI and (ϕ, ψ) = (−75°, 145°) for PPII.52 PPI structure is relatively compact having a helical pitch of 5.6 Å/turn and 3.3 residues/turn53 while PPII helix is highly extended with a helical pitch of 9.3 Å/turn and 3 residues/turn.54 Solvent conditions govern polyproline to adopt PPI or PPII conformation. PPI only forms in some specific organic solvents, such as n-propanol, whereas PPII dominates in aqueous solution.55−57 Given that polyproline can adopt either all cis PPI or all trans PPII conformation, it has been a valuable model for studying the cis−trans isomerizations of amide bonds.17,58 Recently, Zondlo and co-workers have prepared a series of tetrapeptides and octapeptides, and applied NMR and CD spectroscopy to demonstrate that aromatic−proline interactions could form in a polyproline helix by incorporating an aromatic residue into the middle of polyproline sequence and modulate the population of cis prolyl bonds via tuning the electronics of aromatic ring.8,59 Although these aforementioned studies have shown the importance of aromatic−proline interactions on peptide structures and PPII conformation, no work examining the impacts of aromatic−proline interactions on PPI helices and the conversion kinetics between PPI and PPII has been reported. In particular, the temperature-dependent kinetics on the conversion of PPI to PPII has never been studied for aromatic containing oligoprolines. In addition, Wennemers and coworkers showed a position dependent effect on polyproline conformation, in which the same functional group at the C- or Nterminus could lead to different conformational stabilities on PPII.60 Our recent study also demonstrated that terminal stereoelectronic effects on PPII stability are directional.61 Accordingly, here we intended to study terminal aromatic− proline effects, including aromatic-cis-proline interactions at the N-terminus and cis-proline−aromatic interactions at the Cterminus, on polyproline conformation and the kinetics of PPI → PPII conversion. In this work, we designed and synthesized a

series of polyproline peptides with aromatic residues (W, Y, and F) incorporated into the N-terminal or the C-terminal end as shown below. P12: NH−(Pro)12 −OH

N-Terminal Aromatic Peptides. FP11: NH 2−Phe−(Pro)11−OH YP11: NH 2−Tyr−(Pro)11−OH

WP11: NH 2−Trp−(Pro)11−OH

C-Terminal Aromatic Peptides. P11F: NH−(Pro)11−Phe−OH P11Y: NH−(Pro)11−Tyr−OH P11W: NH−(Pro)11−Trp−OH

By CD spectroscopy, we studied the thermodynamic and kinetic consequences of incorporating terminal aromatic residues on polyproline structure. The experimental results revealed that the N-terminal aromatic−proline (aromatic-cis-proline) interaction induces a larger impact than the C-terminal aromatic− proline (cis-proline−aromatic) interaction on PPI forming propensity, and the effect is correlated with the aromatic electronics. Although the results showed that aromatic−proline interactions do not have an important contribution to the kinetics of PPI → PPII conversion, we found that the activation enthalpy and entropy of PPI → PPII transition are highly correlated with the hydrophobicity of aromatic side chains. Together, this work provides a piece of new and valuable information on polyproline folding.

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MATERIALS AND METHODS General Data. Reagents and amino acids were obtained from Aldrich-Sigma Chemical, Alfa, Aesar, Fluka, Novabiochem, or Advanced ChemTech and used without further purification. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of all peptides were obtained using an Autoflex III Smartbeam LRF200-CID spectrometer (Bruker Daltonics). Attachment of Fmoc-Pro-OH, Fmoc-Phe-OH, FmocTyr-OH, and Fmoc-Trp-OH to 2-Chlorotrityl Resin. A solution of Fmoc-protected amino acid (0.2 mmol) and N,Ndiisopropylethylamine (DIEA) (0.13 mL, 0.60 mmol) in dry CH2Cl2 (1.5 mL) was added to another solution in which 0.20 mmol 2-chlorotrityl resin (200 mg, loading capacity 1.0 mmol g−1) were swelled in dry CH2Cl2 (3.0 mL) under N2(g). The mixture was stirred under N2(g) at room temperature for 2 h, and then was added anhydrous CH3OH (2.5 mL) to cap any remaining active sites on the resin. The resin-bound Fmocprotected amino acid was isolated by filtration, washed with dry CH2Cl2 (∼30 mL), and dried under reduced pressure. Loading capacity was measured by UV spectroscopy using the reported protocol (Applied Biosystems Technical Note 123485, Rev. 2, http://www3.appliedbiosystems.com/sup/gl/search.htm) to be 0.63 mmol g−1 for Fmoc-Pro-resin, 0.63 mmol g−1 for Fmoc-Pheresin, 0.71 mmol g−1 for Fmoc-Tyr-resin, and 0.52 mmol g−1 for Fmoc-Trp-resin. Peptide Synthesis and Purification. All of the peptides were prepared on a 0.05 mmol scale using standard solid-phase methods and Fmoc-chemistry protocols. O-BenzotriazoleB


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Figure 2. Far-UV CD spectra for (A) P12 and N-terminal aromatic peptides and (B) P12 and C-terminal aromatic peptides in pH 7.0 and 20 mM sodium phosphate buffer at 25 °C.

N,N,N′,N′-tetramethyluronium (HBTU)-mediated coupling reactions were carried out on an automated PS3 peptide synthesizer (Protein Technologies Inc.). Use of preloaded 2chlorotrityl resin generated a free C-terminus upon cleavage by the solution of 95% (v/v) trifluoroacetic acid (TFA), 2.5% triisopropylsilane, and 2.5% H2O. Each peptide also has a free N terminus. All peptides were purified by reverse-phase highperformance liquid chromatography (HPLC) with a Vydac semipreparative C18-column. The H2O/acetonitrile gradients with 0.1% (w/v) TFA were used as the eluting solvent system to purify the peptides. According to HPLC analysis, each purified peptide was more than 90% pure. Identities of all the peptides were confirmed by MALDI-TOF mass spectrometry and their calculated and observed molecular weights are shown in Table S1 of the Supporting Information. Circular Dichroism (CD) Spectroscopy. All CD measurements were conducted on an Aviv Model 410 circular dichroism spectrometer. Far-UV CD spectra were obtained at 25 °C in pH 7.0 and 20 mM sodium phosphate buffer or in 95% (v/v) npropanol using a 1 mm quartz cuvette and a spectrometer bandwidth of 1 nm. All the peptides in 95% (v/v) n-propanol were incubated for more than 4 days before measurements to allow formation of PPI helices. The peptide concentration was 50−150 μM for the measurements. The concentration of Tyrcontaining peptides or Trp-containing peptides was determined by the UV absorbance at 280 nm in 6 M guanidine hydrochloride at pH 6.5, using an absorption coefficient of 1490 M−1 cm−1 for Tyr and 5500 M−1 cm−1 for Trp. The concentration of Phecontaining peptides was determined by the UV absorbance at 257 nm in 6 M guanidine hydrochloride at pH 6.5, using an absorption coefficient of 200 M−1 cm−1. Because of lack of aromatic residues in P12, the peptide concentration was determined using the UV absorbance (A205) at 205 nm,62,63 and the equation, c (mol L−1) = A205/(31 × l × Mw), where l is the path length of the cell and Mw is the molar mass of the peptide.

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band between 220 and 230 nm and a negative band between 200 and 210 nm, indicating that all of the peptides form PPII helices in aqueous solution. Compared to P12, Tyr-substituted and Trpsubstituted peptides display a more intense positive molar ellipticity at around 229 nm, while Phe-substituted peptides have a weaker one in this region (Table 1). The results are similar to Table 1. Circular Dichroism Parameters for Peptides at 25 °C pH 7.0 and 20 mM phosphate buffer

95% (v/v) n-propanol

peptide

λmax (nm)

[θ]max (103 deg cm2 dmol−1)

λmax (nm)

[θ]max (104 deg cm2 dmol−1)

P12 FP11 YP11 WP11 P11F P11Y P11W

229 229 229 227 229 229 229

1.42 1.41 2.74 5.25 0.66 2.52 2.62

214 214 214 214 214 214 214

2.14 2.71 4.73 3.81 2.53 3.52 3.45

the observations by Zondlo et al.8,59 that incorporating Tyr or Trp into a polyproline peptide induced strong positive CD signals between 220 and 230 nm. From previous studies, it has been demonstrated that aromatic residues would induce a strong CD signal around 220 nm due to the electronic absorption of aromatic chromophores.64−66 The signal contribution from aromatic residues may overlap the positive band of PPII conformation, and thus, the molar ellipticity within the range of 220 to 230 nm is not an appropriate index for the comparison of PPII stability or content in solution. Therefore, the effects of terminal aromatic−proline interactions on PPII forming propensity could not be directly distinguished in these peptides. CD spectra were also recorded for the samples in n-propanol. As shown in Figure 3, the far-UV CD spectra for all the peptides in 95% (v/v) n-propanol display the characteristics of PPI conformation with a strong positive band at 214 nm, showing that all the aromatic-substituted peptides form PPI helices under this condition. The positive value of molar ellipticity in the region of 210 to 220 nm was usually used as an indicator of PPI contents. In comparison with P12, the aromatic-substituted peptides have a more intense molar ellipticity at 214 nm, indicating that aromatic peptides favor PPI conformation in 95% (v/v) npropanol. The results also suggest that terminal aromatic−

RESULTS AND DISCUSSION

Effects of the Terminal Aromatic Residues on PPI And PPII Forming Propensity. CD spectroscopy was used to characterize the peptide structures in solution. As shown in Figure 2, all the peptides in phosphate buffer exhibit a similar farUV CD spectrum attributed to PPII conformation with a positive C


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Figure 3. Far-UV CD spectra for (A) P12 and N-terminal aromatic peptides; (B) P12 and C-terminal aromatic peptides in 95% (v/v) n-propanol at 25 °C.

propanol at 25 °C. Upon diluting the sample that was preincubated in n-propanol and mainly contained PPI helices with phosphate buffer into 90% (v/v) aqueous solution, a series of kinetic CD spectra were recorded. As shown in Figure 4, all the peptides exhibit a transition from PPI conformation (with a strong positive peak 214 nm) to PPII conformation (with a positive band between 220 and 230 nm and a negative band between 200 and 210 nm) with an isosbestic point approximately at 225 nm as time evolved. The observation of an isosbestic point indicates that only two conformations, PPI and PPII, exist in the conversion process, similar to what was found in our previous studies.61,67 Because the conformation of polyproline was controlled by solvent condition, we assumed that PPI → PPII conversion kinetics follows an irreversible process and a first-order kinetics. The relationship can be written as follows,

proline interactions may occur and enhance PPI contents in npropanol. In contrast to PPII helices, the terminal aromatic residues seem to impose a direct and distinct effect on PPI conformation. As shown in Table 1, we found that among the Nterminal aromatic peptides, YP11 has the largest positive maximum (4.73 × 104 deg cm2 dmol−1), followed by WP11 (3.81 × 104 deg cm2 dmol−1) and FP11 (2.71 × 104 deg cm2 dmol−1). The intensity in CD signals at 214 nm for the Cterminal aromatic-substituted peptides also follows a similar order: P11Y (3.52 × 104 deg cm2 dmol−1), P11W (3.45 × 104 deg cm2 dmol−1), and P11F (2.53 × 104 deg cm2 dmol−1). The CD measurements indicate that the Tyr-Pro pair induces the largest increase in PPI contents while the Phe-Pro pair has the least effect on PPI contents, concurring with the previous finding that the Tyr-Pro and Trp-Pro interactions could induce the population of cis peptide bonds more than the Phe-Pro interaction.4,15−18 Although the previous work on short peptides showed that Trp-Pro generated a stronger aromatic−proline interaction than Tyr-Pro because the sequence of Trp-Pro had a higher percent of cis peptide bonds,4,8 the stronger interaction between Pro and Trp did not completely reflect on PPI contents in n-propanol. This might be due to the steric effects from the bulky side chain of Trp, which prevent Trp from forming a strong interaction with Pro within a long polyproline peptide. More interestingly, we found that the same aromatic residue had a different effect from the N- and C-termini of polyproline, in which the substitution at the N-terminus generated a more significant impact on PPI structure than that at the C-terminus. The observation of terminal effects implies that the aromatic−cisproline configuration may form a stronger interaction than cisproline−aromatic configuration to induce a greater PPI forming propensity in 95% (v/v) n-propanol. Impact of the Terminal Aromatic Residues on PPI → PPII Conversion Kinetics. From CD measurements, we could not observe a distinct effect imposed by various aromatic amino acids on PPII conformation. In order to gain more insights into the consequences of incorporating aromatic residues into the polyproline termini, we performed the measurements of PPI → PPII conversion kinetics. Since PPII helices are predominated in aqueous solution and PPI helices are favored in n-propanol, we used a solvent-switching method as described in our previous studies61,67 to measure the PPI → PPII conversion rate in a solution of 90% (v/v) sodium phosphate buffer and 10% (v/v) n-

k

PPI → PPII

The concentration of PPI and PPII changes with time (t) and can thus be written as [PPI]t = [PPI]0 e−kt

(1)

[PPII]t = [PPI]0 (1 − e−kt )

(2)

where [PPI]0 denotes the initial concentration of PPI and k is the rate constant. Because the ellipticity possesses the proportionality of the concentration, similar to absorbance,68 the temporal profiles of the ellipticity was employed to illustrate the evolutions of PPI and PPII. The time dependent CD signal θt which is composed of the molar ellipticity from PPI (θI) and PPII (θII) can be described as θt = θI[PPI]t + θII[PPII]t = θI[PPI]0 e−kt + θII[PPI]0 (1 − e−kt )

(3)

To simplify the equation, the initial CD signal (θ0) was subtracted from θt and eq 3 becomes θt − θ0 = [θI[PPI]0 e−kt + θII[PPI]0 (1 − e−kt )] − θI[PPI]0 = (θII[PPI]0 − θI[PPI]0 )(1 − e−kt ) (4) D


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Figure 4. Representative time-dependent CD spectra for P12 and aromatic peptides in 90% (v/v) aqueous solution at 25 °C.

Plotting the PPI signal difference (θt − θ0) at 214 nm versus time shows a single exponential decay for each peptide (Figure 5). The PPI → PPII conversion rate constant was determined by fitting the data into a first-order exponential decay eq 5,

θt − θ0 = a(1 − e−kt )

(5)

As shown in Table 2, all the aromatic peptides have a larger rate constant than that of P12 at 25 °C, showing that the incorporation of an aromatic residue into the termini can induce E


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Figure 5. Plots of the difference between the maximum PPI signals (214 nm) at time t and time zero (t0) versus time for P12 and aromatic peptides in 90% (v/v) aqueous solution at 25 °C. The solid lines represent the best fit to a single exponential decay.

a faster PPI → PPII transition rate than P12 (kP12 = 1.95 × 10−4 s−1 at 25 °C). The replacement of Pro to different amino acids speeding the conversion of PPI to PPII was also observed in the previous studies.61,67

To investigate the activation energy of PPI → PPII conversion, we further performed the kinetic study at various temperatures. Therefore, a series of time-dependent CD spectra were recorded for each of the peptides at three other temperatures (4, 15, 30 F


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Table 2. Rate Constants of PPI → PPII Conversion at Different Temperatures for the Peptides in 90% (v/v) Aqueous Solution PPI → PPII rate constant (s−1)a

a

peptide

4 °C

15 °C

25 °C

30 °C

P12 FP11 YP11 WP11 P11F P11Y P11W

6.26 (0.44) × 10−6 1.89 (0.62) × 10−5 1.09 (0.19) × 10−5 1.89 (0.28) × 10−5 1.54 (0.40) × 10−5 7.08 (0.34) × 10−6 1.45 (0.50) × 10−5

5.12 (0.24) × 10−5 7.55 (0.15) × 10−5 8.09 (0.10) × 10−5 7.45 (0.15) × 10−5 5.94 (0.18) × 10−5 6.08 (0.17) × 10−5 5.86 (0.26) × 10−5

1.95 (0.05) × 10−4 2.72 (0.03) × 10−4 2.73 (0.02) × 10−4 2.58 (0.03) × 10−4 2.02 (0.10) × 10−4 2.10 (0.05) × 10−4 2.11 (0.05) × 10−4

3.47 (0.09) × 10−4 4.66 (0.08) × 10−4 4.91 (0.05) × 10−4 4.40 (0.08) × 10−4 3.64 (0.09) × 10−4 4.00 (0.05) × 10−4 3.72 (0.06) × 10−4

The values in parentheses represent the standard errors of the fitting.

Figure 6. Representative time-dependent CD spectra for FP11 in 90% (v/v) aqueous solution at (A) 4 °C, (B) 15 °C, (C) 25 °C, and (D) 30 °C.

°C), and these spectra were shown in the Supporting Information (Figures S1, S3, and S5). Taking FP11 peptide as an example, the isosbestic point was found at each temperature measured (Figure 6), indicating that the PPI → PPII conversion process is similar at different temperatures. From the decay curves (Figures S2, S4, and S6 in the Supporting Information), the rate constants at different temperatures were determined. As shown in Table 2, the rate of PPI → PPII conversion becomes faster upon increasing the temperature. This finding is similar to a previous study that observed a temperature-dependent PPI → PPII conversion rate from the analysis of temperature-induced PPI → PPII transition hysteresis curves.69 A plot of ln(k/T) versus 1/T displays a linear relation for each peptide (Figure 7). The activation enthalpy (ΔH‡) and activation entropy (ΔS‡) can be obtained by fitting the data into the Eyring equation, eq 6, ln

k k ΔH ‡ 1 ΔS‡ =− × + ln B + T R T h R

where k is rate constant, T is absolute temperature, R is gas constant, kB is Boltzmann’s constant, and h is Planck’s constant. The determined ΔH‡ and ΔS‡ were employed to derive the differences in free energy of activation (ΔΔG‡) between P12 and aromatic peptides at 25 °C as listed in Table 3. At 25 °C all the aromatic peptides have similar values of activation free energy, which are lower than that of P12, indicating that they have a faster PPI → PPII conversion rate than P12. In addition, the conversion rate and activation free energy only slightly differ for a same aromatic substitution at the C-terminus and the N-terminus, suggesting that the effects on PPI → PPII transition kinetics upon incorporating an aromatic residue into the terminal position are nondirectional. Surprisingly, as shown in Figure 7, we found that the Phecontaining and Trp-containing peptides exhibit an almost identical plot while the linear plots of the Tyr-containing peptides and P12 possess a similar slope. The results indicate that Tyr affects the PPI to PPII conversion kinetics differently from Phe and Trp. The Phe-containing and Trp-containing peptides

(6) G


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Compared to P12, the Tyr-containing peptides only have a small difference in activation enthalpy (ΔΔH‡ = −6.0 and −0.4 kJ mol−1) and activation entropy (ΔΔS‡ = −17.2 and −0.2 J mol−1 K−1), indicating that the kinetics of PPI → PPII conversion for the Tyr-containing peptides is relatively similar to that for P12. In contrast, for the Phe-containing and Trp-containing peptides, their ΔH‡ and ΔS‡ values are dramatically different from the values of P12 and the Tyr-containing peptides, where the ΔΔH‡ and ΔΔS‡ values are approximately −20 kJ mol−1 and −70 J mol−1 K−1. We could rationalize the results by considering the side chain hydrophobicity of these three aromatic residues and proline. The side chain hydrophobicity of these amino acid is in the order of Trp > Phe > Tyr > Pro,70,71 which coincides with the differences in activation entropy and enthalpy observed between P12 and aromatic peptides. The side chain hydrophobicity of these amino acids defined by the free energy of transferring from water to alcohol (ΔGwater→alcohol) at 25 °C was previously reported as the following: Pro −4.18 kJ mol−1, Tyr −5.43 kJ mol−1, Phe −10.0 kJ mol−1, and Trp −12.5 kJ mol−1.72 Compared to P12, a large difference in hydrophobicity between an aromatic residue and Pro will lead to a pronounced effect on the activation enthalpy and entropy of PPI → PPII for its corresponding peptide. Thus, the more hydrophobic Phe and Trp cause a larger effect than the less hydrophobic Tyr. Because the hydrophobicity of Tyr is relatively similar to that of Pro, the Tyr-containing peptides behave more like P12 while converting PPI to PPII conformation. Our analysis shows that P12, YP11, and P11Y have larger activation enthalpies than FP11, P11F, WP11, and P11W, suggesting that the more hydrophilic Pro and Tyr might make their corresponding peptides need more energy to rearrange their interactions with water and the hydrogen bonding network within water matrix when they are first transferred to aqueous solution and start the transformation of PPI to PPII. A recent report showed that a more hydrophobic proline derivative has a large dipole moment difference (Δμcis−trans) between cis and trans conformers and has a greater isomerism equilibrium constant Ktrans/cis in a less polar solvent.73 From this aspect, we could also rationalize our results in terms of Δμcis−trans and Ktrans/cis as follows. The more hydrophobic Phe and Trp residues could make the polyproline peptides more populated in trans conformers in less polar n-propanol and thus require less enthalpy to activate the conversion of PPI to PPII in aqueous solution. In contrast, the less hydrophobic Tyr and Pro residues with a smaller Δμcis−trans do not facilitate the polyproline peptides to form a respectable amount of trans conformers in n-propanol, leading to the requirement of a larger activation enthalpy while converting to PPII in aqueous solution. The relative hydrophobicity of aromatic side-chains also reflects

Figure 7. Plots of ln(k/T) versus 1/T for (A) P12 and N-terminal aromatic peptides, and (B) P12 and C-terminal aromatic peptides in 90% (v/v) aqueous solution. The lines represent the best fit to Eyring equation.

have a similar activation enthalpy (82.5−85.2 kJ mol−1), which is much smaller than that of P12 (105.2 kJ mol−1) and the Tyrcontaining peptides (99.2 and 104.8 kJ mol−1), suggesting that aromatic−proline interactions may be not a key factor in activating this structural transformation. The data show that Tyrsubstitutions introduce a different effect on the activation enthalpy and entropy of PPI → PPII transition from Phe- and Trp-substitutions.

Table 3. Activation Parameters of PPI → PPII Conversion for the Peptides in 90% (v/v) Aqueous Solution peptide

ΔH‡ (kJ mol−1)

ΔS‡ (J mol−1 K−1)

ΔΔH‡ a (kJ mol−1)

ΔΔS‡ b (J mol−1 K−1)

ΔΔG‡ c (25 °C) (kJ mol−1)

P12 FP11 YP11 WP11 P11F P11Y P11W

105.2 (6.4)d 84.2 (1.2) 99.2 (6.3) 82.5 (0.9) 82.6 (1.1) 104.8 (7.2) 85.2 (1.1)

36.8 (22.0) −30.9 (4.2) 19.6 (21.5) −37.0 (3.2) −38.5 (3.9) 36.6 (24.7) −29.6 (3.7)

− −21.0 −6.0 −22.7 −22.6 −0.4 −20.0

− −67.7 −17.2 −73.8 −75.3 −0.2 −66.4

− −0.83 −0.87 −0.71 −0.16 −0.34 −0.21

a ΔΔH‡ = ΔH‡(aromatic peptide) − ΔH‡(P12). bΔΔS‡ = ΔS‡(aromatic peptide) − ΔS‡(P12). cΔΔG‡ = ΔΔH‡ − TΔΔS‡; T is 298 K. dThe values in parentheses represent the standard errors of the fitting.

H


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the National Center for High-Performance Computing (NCHC) for computer time and facilities.

on the impacts of activation entropy. For Phe- and Trpcontaining peptides, the more hydrophobic side chains of Phe and Trp would largely restrict the freedom of water molecules when a compact PPI helix converts to an extended PPII helix to cause the side-chains more exposed to water, leading to a decrease in entropy. To the best of our knowledge, this is the first study to report the relationship between the aromatic side chain hydrophobicity and the conversion kinetics of PPI to PPII using such a temperature-dependent kinetics approach.

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(1) Creighton, T. E. Proteins: Structures and Molecular Properties. W. H. Freeman and Company: New York, 1993; p 507. (2) Brandts, J. F.; Halvorson, H. R.; Brennan, M. Consideration of the possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. Biochemistry 1975, 14, 4953− 4963. (3) Stewart, D. E.; Sarkar, A.; Wampler, J. E. Occurrence and role of cis peptide bonds in protein structures. J. Mol. Biol. 1990, 214, 253−260. (4) Zondlo, N. J. Aromatic−proline interactions: Electronically tunable CH/π interactions. Acc. Chem. Res. 2013, 46, 1039−1049. (5) Ma, B.; Elkayam, T.; Wolfson, H.; Nussinov, R. Protein−protein interactions: Structurally conserved residues distinguish between binding sites and exposed protein surfaces. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 5772−5777. (6) Vermeulen, W.; Van Troys, M.; Bourry, D.; Dewitte, D.; Rossenu, S.; Goethals, M.; Borremans, F. A. M.; Vandekerckhove, J.; Martins, J. C.; Ampe, C. Identification of the PXW sequence as a structural gatekeeper of the headpiece C-terminal subdomain fold. J. Mol. Biol. 2006, 359, 1277−1292. (7) Bhattacharyya, R.; Chakrabarti, P. Stereospecific interactions of proline residues in protein structures and complexes. J. Mol. Biol. 2003, 331, 925−940. (8) Pandey, A. K.; Thomas, K. M.; Forbes, C. R.; Zondlo, N. J. Tunable control of polyproline helix (PPII) structure via aromatic electronic effects: An electronic switch of polyproline helix. Biochemistry 2014, 53, 5307−5314. (9) Neidigh, J. W.; Fesinmeyer, R. M.; Andersen, N. H. Designing a 20residue protein. Nat. Struct. Biol. 2002, 9, 425−430. (10) Xiao, S. F.; Bi, Y.; Shan, B.; Raleigh, D. P. Analysis of core packing in a cooperatively folded miniature protein: The ultrafast folding villin headpiece helical subdomain. Biochemistry 2009, 48, 4607−4616. (11) Zheng, T.-Y.; Lin, Y.-J.; Horng, J.-C. Thermodynamic consequences of incorporating 4-substituted proline derivatives into a small helical protein. Biochemistry 2010, 49, 4255−4263. (12) London, N.; Movshovitz-Attias, D.; Schueler-Furman, O. The structural basis of peptide-protein binding strategies. Structure 2010, 18, 188−199. (13) Glaser, F.; Steinberg, D. M.; Vakser, I. A.; Ben-Tal, N. Residue frequencies and pairing preferences at protein−protein interfaces. Proteins: Struct., Funct., Genet. 2001, 43, 89−102. (14) Brandl, M.; Weiss, M. S.; Jabs, A.; Sühnel, J.; Hilgenfeld, R. CH···π-interactions in proteins. J. Mol. Biol. 2001, 307, 357−377. (15) Thomas, K. M.; Naduthambi, D.; Zondlo, N. J. Electronic control of amide cis−trans isomerism via the aromatic−prolyl interaction. J. Am. Chem. Soc. 2006, 128, 2216−2217. (16) Yao, J.; Dyson, H. J.; Wright, P. E. Three-dimensional structure of a type VI turn in a linear peptide in water solution Evidence for stacking of aromatic rings as a major stabilizing factor. J. Mol. Biol. 1994, 243, 754−766. (17) Fischer, G. Chemical aspects of peptide bond isomerisation. Chem. Soc. Rev. 2000, 29, 119−127. (18) Wu, W.-J.; Raleigh, D. P. Local control of peptide conformation: Stabilization of cis proline peptide bonds by aromatic proline interactions. Biopolymers 1998, 45, 381−394. (19) Ferreon, J. C.; Hilser, V. J. The effect of the polyproline II (PPII) conformation on the denatured state entropy. Protein Sci. 2003, 12, 447−457. (20) Hamburger, J. B.; Ferreon, J. C.; Whitten, S. T.; Hilser, V. J. Thermodynamic mechanism and consequences of the polyproline II (PII) structural bias in the denatured states of proteins. Biochemistry 2004, 43, 9790−9799. (21) Whittington, S. J.; Chellgren, B. W.; Hermann, V. M.; Creamer, T. P. Urea promotes polyproline II helix formation: implications for protein denatured states. Biochemistry 2005, 44, 6269−6275.

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CONCLUSIONS In this work, we have used aromatic-substituted polyproline peptides to demonstrate that aromatic−proline interactions significantly affect the forming propensity of PPI helices, and the consequences essentially depend on the position in a peptide and the interacting pairs. The N-terminal aromatic−proline interaction (aromatic-cis-Pro) induces a greater PPI-forming propensity than the C-terminal aromatic−proline interaction (cis-Pro-aromatic). The tendency of PPI contents in solution is essentially related with the strength of aromatic−proline interactions in the order of Pro-Tyr > Pro-Trp > Pro-Phe. For the PPI → PPII conversion kinetics, instead of aromatic−proline interactions, the hydrophobicity of aromatic side chains plays a critical role in activating this process. Although aromatic−proline interactions in proteins and peptides have been widely studied in the past decades, this work reports the first study of terminal aromatic−proline effects on polyproline conformation, in particular the impacts on PPI contents in solution. Additionally, using a temperature-dependent approach to investigate the effects of aromatic residues on the kinetics of PPI → PPII conversion discovers new valuable information about the impacts of aromatic side chain hydrophobicity on the transformation of PPI to PPII, which has never been reported. The current work reveals news insight into the effects of aromatic-substitution on PPI forming propensity and the kinetics of PPI → PPII conversion, allowing us to have a better understanding on what roles an aromatic residue play on the conformational stability and transformation of a polyproline helix. Our approach would also provide a useful strategy for further quantitative studies of polyproline folding.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b08717. Table listing the measured molecular weights for the peptides, additional time-dependent CD spectra at different temperatures, and plots of maximal CD signals versus time are included. (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*(J.C.-H.) Telephone: +886-3-5715131 ext 35635. Fax: +886-35711082. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thankfully acknowledge financial support from the Ministry of Science and Technology, Taiwan (Grants: MOST 103-2113M-007-008 and NSC 98-2119-M-007-011) and National Tsing Hua University (Grant: 104N2011E1). We are also grateful to I


Article

The Journal of Physical Chemistry B (22) Deutch, C. E.; Arballo, M. E.; Cooks, L. N.; Gomes, J. M.; Williams, T. M.; Aboul-Fadl, T.; Roberts, J. C. Susceptibility of Escherichia coli to L-selenaproline and other L-proline analogues in laboratory culture media and normal human urine. Lett. Appl. Microbiol. 2006, 43, 392−398. (23) Wedemeyer, W. J.; Welker, E.; Scheraga, H. A. Proline cis−trans isomerization and protein folding. Biochemistry 2002, 41, 14637−14644. (24) Siligardi, G.; Drake, A. F. The importance of extended conformations and, in particular, the PII conformation for the molecular recognition of peptides. Biopolymers 1995, 37, 281−292. (25) Vanhoof, G.; Goossens, F.; De Meester, I.; Hendriks, D.; Scharpe, S. Proline motifs in peptides and their biological processing. FASEB J. 1995, 9, 736−744. (26) Kleywegt, G. J.; Jones, T. A. Phi/psi-chology: Ramachandran revisited. Structure 1996, 4, 1395−1400. (27) Brandsch, M.; Thunecke, F.; Küllertz, G.; Schutkowski, M.; Fischer, G.; Neubert, K. Evidence for the absolute conformational specificity of the intestinal H+/peptide symporter, PEPT1. J. Biol. Chem. 1998, 273, 3861−3864. (28) Sarkar, P.; Reichman, C.; Saleh, T.; Birge, R. B.; Kalodimos, C. G. Proline cis-trans isomerization controls autoinhibition of a signaling protein. Mol. Cell 2007, 25, 413−426. (29) Zarrinpar, A.; Bhattacharyya, R. P.; Lim, W. A. The structure and function of proline recognition domains. Sci. Signaling 2003, 2003, re8− re8. (30) Adzhubei, A. A.; Sternberg, M. J. E.; Makarov, A. A. Polyproline-II helix in proteins: Structure and function. J. Mol. Biol. 2013, 425, 2100− 2132. (31) Rath, A.; Davidson, A. R.; Deber, C. M. The structure of “unstructured” regions in peptides and proteins: Role of the polyproline II helix in protein folding and recognition. Biopolymers 2005, 80, 179− 185. (32) Hu, K.-N.; Havlin, R. H.; Yau, W.-M.; Tycko, R. Quantitative determination of site-specific conformational distributions in an unfolded protein by solid-state nuclear magnetic resonance. J. Mol. Biol. 2009, 392, 1055−1073. (33) Blanch, E. W.; Morozova-Roche, L. A.; Cochran, D. A. E.; Doig, A. J.; Hecht, L.; Barron, L. D. Is polyproline II helix the killer conformation? A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme. J. Mol. Biol. 2000, 301, 553−563. (34) Ishijima, J.; Nagasaki, N.; Maeshima, M.; Miyano, M. RVCaB, a calcium-binding protein in radish vacuoles, is predominantly an unstructured protein with a polyproline type II helix. J. Biochem. 2007, 142, 201−211. (35) Arora, P. S.; Ansari, A. Z.; Best, T. P.; Ptashne, M.; Dervan, P. B. Design of artificial transcriptional activators with rigid poly-L-proline linkers. J. Am. Chem. Soc. 2002, 124, 13067−13071. (36) Bonger, K. M.; Kapoerchan, V. V.; Grotenbreg, G. M.; van Koppen, C. J.; Timmers, C. M.; van der Marel, G. A.; Overkleeft, H. S. Oligoproline helices as structurally defined scaffolds for oligomeric G protein-coupled receptor ligands. Org. Biomol. Chem. 2010, 8, 1881− 1884. (37) Cordes, M.; Koettgen, A.; Jasper, C.; Jacques, O.; Boudebous, H.; Giese, B. Influence of amino acid side chains on long-distance electron transfer in peptides: Electron hopping via ″Stepping Stones″. Angew. Chem., Int. Ed. 2008, 47, 3461−3463. (38) Fillon, Y. A.; Anderson, J. P.; Chmielewski, J. Cell penetrating agents based on a polyproline helix scaffold. J. Am. Chem. Soc. 2005, 127, 11798−11803. (39) Garbuio, L.; Lewandowski, B.; Wilhelm, P.; Ziegler, L.; Yulikov, M.; Wennemers, H.; Jeschke, G. Shape persistence of polyproline II helical oligoprolines. Chem. - Eur. J. 2015, 21, 10747−10753. (40) Geisler, I.; Chmielewski, J. Probing length effects and mechanism of cell penetrating agents mounted on a polyproline helix scaffold. Bioorg. Med. Chem. Lett. 2007, 17, 2765−2768. (41) Haas, E.; Wilchek, M.; Katchalskikatzir, E.; Steinberg, I. Z. Distribution of end-to-end distances of oligopeptides in solution as estimated by energy-transfer. Proc. Natl. Acad. Sci. U. S. A. 1975, 72, 1807−1811.

(42) Kroll, C.; Mansi, R.; Braun, F.; Dobitz, S.; Maecke, H. R.; Wennemers, H. Hybrid bombesin analogues: Combining an agonist and an antagonist in defined distances for optimized tumor targeting. J. Am. Chem. Soc. 2013, 135, 16793−16796. (43) Lewandowska, U.; Zajaczkowski, W.; Chen, L.; Bouilliere, F.; Wang, D.; Koynov, K.; Pisula, W.; Muellen, K.; Wennemers, H. Hierarchical supramolecular assembly of sterically demanding π-systems by conjugation with oligoprolines. Angew. Chem., Int. Ed. 2014, 53, 12537−12541. (44) Ma, D.; Bettis, S. E.; Hanson, K.; Minakova, M.; Alibabaei, L.; Fondrie, W.; Ryan, D. M.; Papoian, G. A.; Meyer, T. J.; Waters, M. L.; et al. Interfacial energy conversion in Ru-II polypyridyl-derivatized oligoproline assemblies on TiO2. J. Am. Chem. Soc. 2013, 135, 5250− 5253. (45) McCafferty, D. G.; Friesen, D. A.; Danielson, E.; Wall, C. G.; Saderholm, M. J.; Erickson, B. W.; Meyer, T. J. Photochemical energy conversion in a helical oligoproline assembly. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 8200−8204. (46) Sato, S.; Kwon, Y.; Kamisuki, S.; Srivastava, N.; Mao, Q. A.; Kawazoe, Y.; Uesugi, M. Polyproline-rod approach to isolating protein targets of bioactive small molecules: Isolation of a new target of indomethacin. J. Am. Chem. Soc. 2007, 129, 873−880. (47) Schuler, B.; Lipman, E. A.; Steinbach, P. J.; Kumke, M.; Eaton, W. A. Polyproline and the ″spectroscopic ruler″ revisted with singlemolecule fluorescence. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 2754− 2759. (48) Stryer, L.; Haugland, R. P. Energy transfer - a spectroscopic ruler. Proc. Natl. Acad. Sci. U. S. A. 1967, 58, 719−726. (49) Ungar-waron, H.; Gurari, D.; Hurwitz, E.; Sela, M. Role of a rigid polyproline space inserted between hapten and carrier in induction of anti-hapten antibodies and delayed hypersensitivity. Eur. J. Immunol. 1973, 3, 201−205. (50) Upert, G.; Bouillere, F.; Wennemers, H. Oligoprolines as scaffolds for the formation of silver nanoparticles in defined sizes: Correlating molecular and nanoscopic dimensions. Angew. Chem., Int. Ed. 2012, 51, 4231−4234. (51) Wilhelm, P.; Lewandowski, B.; Trapp, N.; Wennemers, H. A crystal structure of an oligoproline PPII-helix, at last. J. Am. Chem. Soc. 2014, 136, 15829−15832. (52) Hopfinger, A. J. Conformational properties of macromolecules; Academic Press: New York, 1973; p 352. (53) Traub, W.; Shmueli, U. Structure of poly-proline I. Nature 1963, 198, 1165−1166. (54) Cowan, P. M.; McGavin, S. Structure of poly-L-proline. Nature 1955, 176, 501−503. (55) Knof, S.; Engel, J. Conformational stability, partial specific volumes and spectroscopic properties of poly-L-proline, poly-Lhydroxyproline and some of its O-acyl-derivatives in various solvent systems. Isr. J. Chem. 1974, 12, 165−177. (56) Mutter, M.; Woehr, T.; Gioria, S.; Keller, M. Pseudo-prolines: Induction of cis/trans-conformational interconversion by decreased transition state barriers. Biopolymers 1999, 51, 121−128. (57) Kakinoki, S.; Hirano, Y.; Oka, M. On the stability of polyproline-I and II structures of proline oligopeptides. Polym. Bull. 2005, 53, 109− 115. (58) Dugave, C.; Demange, L. Cis−trans isomerization of organic molecules and biomolecules: Implications and applications. Chem. Rev. 2003, 103, 2475−2532. (59) Brown, A. M.; Zondlo, N. J. A propensity scale for type II polyproline helices (PPII): Aromatic amino acids in proline-rich sequences strongly disfavor PPII due to proline−aromatic interactions. Biochemistry 2012, 51, 5041−5051. (60) Kuemin, M.; Schweizer, S.; Ochsenfeld, C.; Wennemers, H. Effects of terminal functional groups on the stability of the polyproline II structure: A combined experimental and theoretical study. J. Am. Chem. Soc. 2009, 131, 15474−15482. (61) Lin, Y.-J.; Horng, J.-C. Impacts of terminal (4R)-fluoroproline and (4S)-fluoroproline residues on polyproline conformation. Amino Acids 2014, 46, 2317−2324. J


Article

The Journal of Physical Chemistry B (62) Scopes, R. K. Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 1974, 59, 277−282. (63) Grimsley, G. R.; Pace, C. N. Spectrophotometric Determination of Protein Concentration. In Current Protocols in Protein Science; John Wiley & Sons, Inc.: 2003; pp 3.1.1−3.1.9. (64) Chakrabartty, A.; Kortemme, T.; Padmanabhan, S.; Baldwin, R. L. Aromatic side-chain contribution to far-ultraviolet circular dichroism of helical peptides and its effect on measurement of helix propensities. Biochemistry 1993, 32, 5560−5565. (65) Gilbert, S. M.; Wellner, N.; Belton, P. S.; Greenfield, J. A.; Siligardi, G.; Shewry, P. R.; Tatham, A. S. Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 2000, 1479, 135−146. (66) Bochicchio, B.; Tamburro, A. M. Polyproline II structure in proteins: Identification by chiroptical spectroscopies, stability, and functions. Chirality 2002, 14, 782−792. (67) Chiang, Y.-C.; Lin, Y.-J.; Horng, J.-C. Stereoelectronic effects on the transition barrier of polyproline conformational interconversion. Protein Sci. 2009, 18, 1967−1977. (68) Chiang, H.-K.; Chu, L.-K. Solvent isotope effect on the dark adaptation of bacteriorhodopsin in purple membrane: Viewpoints of kinetics and thermodynamics. J. Phys. Chem. B 2014, 118, 2662−2669. (69) Kuemin, M.; Engel, J.; Wennemers, H. Temperature-induced transition between polyproline I and II helices: quantitative fitting of hysteresis effects. J. Pept. Sci. 2010, 16, 596−600. (70) Mant, C. T.; Kovacs, J. M.; Kim, H.-M.; Pollock, D. D.; Hodges, R. S. Intrinsic amino acid side-chain hydrophilicity/hydrophobicity coefficients determined by reversed-phase high-performance liquid chromatography of model peptides: Comparison with other hydrophilicity/hydrophobicity scales. Biopolymers 2009, 92, 573−595. (71) Roseman, M. A. Hydrophilicity of polar amino acid side-chains is markedly reduced by flanking peptide bonds. J. Mol. Biol. 1988, 200, 513−522. (72) Fauchere, J. L.; Pliska, V. Hydrophobic parameters π of aminoacid side-chains from the partitioning of N-acetyl-amino-acid amides. Eur. J. Med. Chem. 1983, 18, 369−375. (73) Siebler, C.; Maryasin, B.; Kuemin, M.; Erdmann, R. S.; Rigling, C.; Grunenfelder, C.; Ochsenfeld, C.; Wennemers, H. Importance of dipole moments and ambient polarity for the conformation of Xaa-Pro moieties - a combined experimental and theoretical study. Chem. Sci. 2015, 6, 6725−6730.

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Effects of the Terminal Aromatic Residues on Polyproline

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