The Impact of 4-Thiaproline on Polyproline Conformation - The

Article pubs.acs.org/JPCB

The Impact of 4‑Thiaproline on Polyproline Conformation Yu-Ju Lin, Chiao-Hsin Chang, and Jia-Cherng Horng* Department of Chemistry and Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan 30013, R.O.C. S Supporting Information *

ABSTRACT: Proline is unique among the genetically coded amino acids; because of the presence of a saturated pyrrolidine ring, it favors a cis peptide bond more strongly than other amino acids. The prolyl peptide bond conformational preference can be modulated by alterations to the atoms or substitution groups on the ring. In the study of a simple Ac-Xaa-OMe system, (2R)-4-thiaproline (Thp) was shown to favor an endo ring pucker and a cis prolyl peptide bond. Herein, to investigate the effects of Thp on a more complex system, that is, the polyproline structure, we prepared a series of polyproline peptides with one or multiple proline residues substituted with Thp and used circular dichroism (CD) spectroscopy to characterize their structures. In contrast to the results obtained using the Ac-Xaa-OMe system, here we found that Thp not only destabilizes all-trans polyproline II conformation, but also disfavors all-cis polyproline I structure. On the basis of the hybrid density functional theory analysis, we demonstrate that this phenomenon could be due to the small transition barrier between an exo and an endo pucker for the thiazolidine ring of Thp in a PPI helix and a weak backbone n → π* interaction for Thp in PPII conformation. The combination of experimental and computational data allows us to gain new insights into the impact of 4-thiaproline on polyproline conformation.

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INTRODUCTION Among the genetically coded amino acids, only proline has a five-membered ring as a side chain that imposes constraints on the dihedral angles around proline residues. The saturated ring of proline can adopt either a Cγ−exo ring pucker or a Cγ−endo ring pucker in solution, with the Cγ−endo ring pucker being slightly more favorable than the Cγ−exo ring pucker.1−7 Rigidity of the ring also makes X−Pro have the highest propensity to form a cis conformation and limits the available conformations with the ϕ torsion angle restricted to approximately −60° for proline residues. Structural characteristics of the proline ring pucker also correlate with protein backbone conformation in which an exo ring pucker favors trans conformation and a more extended polyproline II (PPII) helix, while an endo ring pucker prefers cis conformation and a more compact polyproline I (PPI) helix.8−12 In addition, proline plays an important role in biologically active peptides or proteins; various proline derivatives can be developed, making alterations to the atoms or substitution groups, to modulate the preference of exo and endo ring puckers, the conformational stability of proteins, and the ligand affinity as well.13−22 The non-natural proline analog (2R)-4-Thiaproline (4thiaproline, Thp) has almost the same number of chemical bonds as that of proline, but its bond lengths of Cβ−S and Cδ− S are longer than those of Cβ−Cγ and Cδ−Cγ in proline while the angle of Cβ−S−Cδ is smaller than that of Cβ−Cγ−Cδ in proline,23−27 indicating that the larger size of sulfur atom causes the geometry of the five-member ring of Thp slightly different © 2014 American Chemical Society

from that of proline. Despite the small difference, Thp can still be considered as isosteric to proline. Its toxicity and effects on the growth and metabolic function of E. coli. K12 have been assessed.28 An early report showed that the incorporation of Thp into human recombinant annexin V destabilized the protein structure slightly but retained protein functions fully.24 Thp was also incorporated into bioactive molecules such as thrombin inhibitors29 and cyclophilin substrates30 to assess its potential use in biological system. One recent study further reported that Thp can be a substrate for human prolyl 4hydroxylase, an enzyme that catalyzes the post-translational hydroxylation of proline residues in protocollagen.31 In addition, Thp has been shown to reduce lipid peroxidation and ventricular collagen accumulation and serve as a substituent of proline in optimizing the pharmacological properties of a protein to make it a potent HIV entry inhibitor.32 The thiazolidine moiety of Thp, regarded as a heterocyclic ring, is also a biologically important scaffold in drug design and known to be associated with several biological activities, including antiviral, antifungal, antihistaminic, hypoglycemic, and antiinflammatory activities.33 An early work showed that the tricyclic thiazolidinlactam-based Pro−Pro mimetics could effectively bind to SH3 domains, suggesting that the thiazolidine moiety might be used to design the nonpeptide Received: April 22, 2014 Revised: August 26, 2014 Published: August 26, 2014 10813

dx.doi.org/10.1021/jp503915p | J. Phys. Chem. B 2014, 118, 10813−10820

The Journal of Physical Chemistry B

Article

ligand of SH3 domains.34 These results strongly suggest that Thp is a potentially good candidate for use in pharmaceutical and drug design. Moreover, both the X-ray crystal structure and the calculation based on the density functional theory (DFT) indicate that the ring side chain of Thp prefers to adopt an endo ring pucker.23,26,35−37 Experimental results further showed that the equilibrium constant between cis and trans conformation (Ktrans/cis) is 2.8 for Thp.35 Although many proline derivatives, in particular 4-substituted derivatives, have been explored to study the consequences of their pucker preference on polyproline conformation,8,10,11,38 studies investigating the effects of Thp on a polyproline helix are relatively rare. The context and features of Thp attract us to investigate its structural role in a polyproline helix and gain more insights into this proline analog. To study the effects of Thp on polyproline conformation, herein we prepared a series of peptides and used circular dichroism (CD) spectroscopy to characterize their structures in different solvents. Experimental results indicate that Thp destabilizes either a PPII helix or a PPI helix. By the DFT analysis, we learned that the energy difference between exo and endo ring puckers for Thp is smaller than that for Pro in PPI structure, leading to a prompt interconversion between these two conformers and affecting the stability of a typical PPI helix. Further, natural bond orbital (NBO) analysis also found that the backbone n → π* interaction in Ac-Thp-OMe is weaker than that in Ac-Pro-OMe, consistent with the experimental observations that Thp-containing peptides form less stable PPII structure. Combining experimental data and computational analysis, here we display that 4-thiaproline, a proline analog, possesses the characteristics that are quite different from those of proline and has a dramatic impact on polyproline conformation.

TFA as the counterion were used to purify the peptides, and all peptides were more than 90% pure according to HPLC analysis. Identities of all peptides were confirmed by MALDITOF (Autoflex III, Bruker Daltonics) mass spectrometry. The calculated and observed molecular masses were as follows: P7 expected 917.47, observed 918.41 [MH+]; Thp7 expected 1043.16, observed 1082.32 [MK+]; P11 expected 1305.68, observed 1306.65 [MH+ ]; Thp-P11 expected 1323.63, observed 1324.81 [MH+]. Circular Dichroism (CD) Spectroscopy. CD measurements were conducted on an Aviv Model 410 CD spectrometer. Far-UV CD spectra were obtained at 4 °C using pH 7.0 and 20 mM sodium phosphate buffer or npropanol. All samples in n-propanol were incubated at 4 °C for at least 4 days before measurements to allow the formation of PPI helices. A peptide concentration of 100 μM was used for measurements. Temperature-induced transition experiments were performed in the temperature from 2 to 94 °C with an interval of 2 °C, and the signals at 214 nm were recorded. Peptide concentrations were determined by the absorbance measurement at 276 nm in 6 M guanidine hydrochloride at pH 6.5, using an absorption coefficient of 1490 M−1 cm−1. Computations. For each model compound and system, hybrid DFT calculations were carried out on all conformations using the Gaussian 09 software in gas phase and at room temperature. For the peptide models, the calculations were also conducted in n-propanol and water. Geometry optimization and frequency calculations were performed at the B3LYP/631+G(d) level of theory. Gibbs free energy and the contributions of enthalpic and entropic terms were obtained at 298 K and l atm. Ea is the activation energy that is the energy difference between the transition state and the ground state. The dihedral angles ϕ, ψ, and χ were measured from the optimized structures for each model compound or peptide. As shown in Scheme 1a, the ϕ is the dihedral angle rotating about

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MATERIALS AND METHODS Peptide Synthesis and Purification. Reagents and amino acids were purchased from Aldrich-Sigma, Alfa Aesar, Fluka, Novabiochem, and Advanced ChemTech, and used without further purification. Fmoc-Tyr-OH was attached to 2chlorotrityl resin following a previously reported method (Applied Biosystems Technical Note 123485, Rev. 2, http:// www3.appliedbiosystems.com/sup/gl/search.htm). All the peptides studied here (see Table 1) were prepared on a 0.05 mmol

Scheme 1. Definition of the Backbone and Pyrrolidine Ring Torsion Angles for Ac-Pro-OMe, and Illustration of a Cγ− endo and a Cγ−exo Pucker

Table 1. Peptides Synthesized in This Study and Their Sequences

a

peptide

sequencea

P11 Thp-P11 P7 Thp7

NH−(Pro)11-Gly-Tyr−OH NH−(Pro)5-Thp-(Pro)5-Gly-Tyr−OH NH−(Pro)7-Gly-Tyr−OH NH−(Thp)7-Gly-Tyr−OH

the N−Cα bond and ψ is the dihedral angle rotating about the Cα−C′ bond, and χ is the dihedral angle N−Cα−Cβ−Cγ. Torsion angles ϕ and ψ determine the secondary structure of polyproline. Torsion angle χ describes the pucker conformation of the pyrrolidine ring, which is positive for an endo pucker (Scheme 1b) and negative for an exo pucker (Scheme 1c).3 NBO39 calculations, as implemented in the Gaussian 09 software, were employed to analyze the optimized structure generated from B3LYP/6-31+G(d) level at the more precise B3LYP/6-311+G(2d,p) level to obtain detailed information about specific orbital information.

Each peptide has free N- and C-termini. Thp is (2R)-4-thiaproline.

scale by solid-phase methods, using Fmoc-protected amino acids and HBTU-mediated coupling reaction on a PS3 synthesizer (Proteins Technologies). The cocktail of 95% trifluoroacetic acid (TFA)/2.5% triisopropylsilane/2.5% H2O was used to cleave the peptide from the resin after completion of peptide syntheses. Upon cleavage, the use of 2-chlorotrityl resin generated a free C-terminus. Peptides were purified by reverse phase HPLC using a Thermo Biobasic semipreparative C18 column. The H2O/acetonitrile gradients with 0.1% (v/v)

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RESULTS AND DISCUSSION As shown in Table 1, we synthesized P11 and Thp-P11 to examine the consequences of substituting one single Pro with 10814



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Figure 1. Far-UV CD spectra for (A) P11, (B) Thp-P11, (C) P7, and (D) Thp7 in pH 7.0 phosphate buffer (closed circles) and n-propanol (open circles) at 4 °C.

Table 2. Circular Dichroism Parameters Measured at 4 °C and Melting Temperatures of PPI Helices for the Peptides n-propanol

pH 7.0 and 20 mM phosphate buffer peptide P11 Thp-P11 P7 Thp7b a

−1

λmax (nm)

[θ]max (deg cm dmol )

λmax (nm)

227 227 226 225

× × × ×

213 216 213 229

2

2.37 1.33 2.33 0.97

3

10 103 103 103

[θ]max (104 deg cm2 dmol−1)

Tm (°C)

× × × ×

52 36

3.20 0.32 1.03 0.29

104 104 104 104

a a

b

Not determined. The CD spectra of Thp7 in aqueous solution and n-propanol also exhibit an intense positive band around 200 nm.

CD signal of Thp-P11 is weaker than that of P11 (1.33 × 103 vs 2.37 × 103 deg cm2 dmol−1), indicating that the replacement of Pro with Thp significantly reduces the PPII content in solution. In n-propanol, P11 displays a typical PPI CD spectrum, in which a strong positive band occurs at around 213 nm and a negative minimum at 197 nm (Figure 1A). For Thp-P11, although its CD spectrum in n-propanol shows the signatures of a PPI helix (Figure 1B), the characteristic CD signal is much weaker than that of P11 (the positive maximum 0.32 × 104 vs 3.2 × 104 deg cm2 dmol−1), suggesting that the PPI content is dramatically reduced for Thp-P11. PPII helices can convert to PPI helices upon changing the solvent from water to n-propanol, and thus one can evaluate the tendency of forming a PPI helix by measuring the fraction of npropanol in solution required for the conversion to occur. Accordingly, we acquired CD spectra for P11 and Thp-P11 at different percentages of n-propanol in solution. As shown in

Thp on the polyproline conformation. The attempt to synthesize (Thp)11-Gly-Tyr peptide was unsuccessful due to difficulty in coupling more than seven Thp residues in a peptide, and low yield, and thus Thp7 was synthesized instead. To compare with Thp7, P7 was also synthesized. The Cterminal Gly-Tyr sequence in each peptide was used for determining its concentration. Effects of Single Thp Substitution on Polyproline Conformation. Because PPII helices normally form in aqueous solution and PPI helices are favored in n-propanol, far-UV CD spectra were recorded in these two solvents for each peptide. As shown in Figure 1A,B, in aqueous solution both P11 and Thp-P11 exhibit a similar far-UV CD spectrum, with a positive band between 220 and 230 nm and a negative minimum around 206 nm, which are characteristics of the PPII conformation, indicating that P11 and Thp-P11 form PPII helices in phosphate buffer. As shown in Table 2, the maximum 10815



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Thp-P11, we performed thermal denaturation of the peptides in n-propanol. As shown in Figure 3, the characteristic CD signals of PPI helices decrease upon heating the samples, indicating that the PPI content of both P11 and Thp-P11 decreases at high temperatures. By taking the first derivatives of the thermal transition curves (Figure S1 in the Supporting Information), we estimated the melting temperatures (Tm) of P11 and Thp-P11 to be 52 and 36 °C respectively. The lower Tm value of ThpP11 again indicates that it forms a less stable PPI structure. Furthermore, by comparing the CD spectra at 4 and 94 °C, we found that in n-propanol Thp-P11 can convert to PPII conformation at high temperatures, while P11 remains in PPI conformation even at a relatively high temperature (Figure S2 in the Supporting Information). These results further highlight the low propensity of Thp to form PPI conformation. Therefore, replacing Pro with Thp not only destabilizes PPII helices but also reduces the tendency to form PPI helices. We further used two more peptides, P7 and Thp7, to investigate the impact of multiple substitutions on polyproline conformation. Similar to P11, P7 also forms PPII structure in aqueous solution and PPI conformation in n-propanol as shown by its CD spectra (Figure 1C). For Thp7, although its CD spectrum in aqueous exhibits the characteristic band of PPII conformation (Figure 1D), the signal is relatively weak in comparison with P7 (0.97 × 103 vs 2.33 × 103 deg cm2 dmol−1), strongly suggesting that the PPII conformation formed by Thp7 is not stable. Surprisingly, Thp7 does not form PPI conformation but remains in PPII structure in npropanol, indicating that Thp7 is highly resistant to the conversion of its PPII conformation to PPI conformation. Observations of the conformations of Thp7 and Thp-P11 demonstrate that the disfavoring effect of Thp on PPI conformation is additive. The finding that Thp7 cannot form PPI helices agrees with the results of early studies that the energy difference between trans and cis forms is very high for the Thp residue, preventing mutarotation from the trans form to the cis form.40,41 CD measurements showed that the substitution of a single proline residue with Thp in the host peptide P11 generates an enormous impact on either PPI or PPII conformation, and Thp exhibits a much lower propensity than Pro to form either PPI or PPII helices, thus reducing the content of either PPI or PPII structures in solution. Such

Figure 2, P11 readily converts from PPII to PPI upon increasing the fraction of n-propanol in solution. In contrast,

Figure 2. Far-UV CD spectra of (A) P11 and (B) Thp-P11 at various percentages of n-propanol in phosphate buffer at 4 °C.

Thp-P11 exhibits a strong resistance to the conversion of PPII to PPI, showing that Thp reduces the propensity to form PPI conformation. The solvent-dependent results are consistent with the far-UV CD measurements. To further examine the thermal stability of the PPI conformation formed from P11 and

Figure 3. Thermal unfolding curves for (A) P11 and (B) Thp-P11 in n-propanol. 10816



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and Thp with trans amide bonds indicate that the endo ring pucker of Thp is distorted and deviates from a regular endo ring pucker as seen in Pro. The significant difference in the relative energy of endo and exo puckers for Thp and Pro in the trans form of Ac-X-OMe may in part explain that the Thpcontaining peptides exhibit a CD spectrum different from that of a regular polyproline helix. We further performed NBO analysis on the trans configurations and exo conformers of Ac-Pro-OMe and AcThp-OMe molecules. The optimized structure and molecular orbital overlapping of Ac-Thp-OMe are illustrated in Figure 5.

impacts could be enhanced by increasing the number of Thp residues in a peptide, as seen in the case of Thp7 peptide.

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COMPUTATIONAL ANALYSIS To have more insights into the effects of Thp on polyproline conformation and rationalize the experimental observations, we first conducted DFT calculations on the conformation of proline and thiaproline. The calculated energy difference between the two ring puckers, Δ(Eendo − Eexo), is shown in Table 3. Pro and Thp prefer an endo pucker in both the trans Table 3. Dihedral Angles and Energy Differences Calculated from the Energy-Optimized Structures of Ac-X-OMe Moleculesa residue (X)

ring pucker

Pro

exo endo exo endo

Thp

Pro Thp a

exo endo exo endo

Δ(Eendo − Eexo) (kcal/mol)

ϕ (deg)

trans Form of Ac-X-OMe −0.32 −59 −70 −0.01 −65 −89 cis Form of Ac-X-OMe −0.53 −64 −78 −0.57 −71 −83

ψ (deg)

χ (deg)

143 152 147 178

−23 31 −27 38

156 161 158 −172

−23 32 −28 42

Calculated in gas phase at B3LYP/6-31+G(d) level of theory.

and cis forms of Ac-X-OMe. In the cis form of Ac-X-OMe, the relative energies for exo- and endo-puckered Thp and Pro are almost identical. It was noted that the ψ-angle of endopuckered Thp shifts to −172° dramatically different from that of endo-puckered Pro, suggesting such an irregular backbone conformation for Thp could impede Thp-containing peptides from forming a typical polyproline structure. In the trans form of Ac-Pro-OMe, the endo conformation is more stable than the exo by 0.32 kcal/mol, consistent with the findings of a previous report by DeRider et al.42 In contrast, in trans Ac-Thp-OMe, the endo conformation is merely more stable than the exo conformation by only 0.01 kcal/mol. Such a small energy difference illustrates that Thp is only slightly biased toward an endo ring pucker and may readily interconvert its ring pucker between endo and exo conformers in the trans configuration. As shown in Figure 4, the energy-minimized conformers of Pro

Figure 5. Illustration of the overlap between the lone pair and π* orbital of Ac-Thp-OMe. The diagram was generated with NBOView 1.1.43

From the optimized structures, we found that the distance (δBD) between Oi‑1 and Ci′ and the Oi‑1... Ci′ = Oi angle (θ) are 2.87 Å and 99.37°, respectively, for Ac-Pro-OMe, and 2.90 Å and 99.58°, respectively, for Ac-Thp-OMe (Table 4). The Table 4. Calculated Energy of Backbone n → π* Interactions for the trans Configurations of Ac-Xaa-OMea residue

ring pucker

δBD (Å)

θ (deg)

stabilization energy (kcal/mol)b

Pro Thp

exo exo

2.87 2.90

99.37 99.98

1.28 1.02

a

Structures were optimized in gas phase at B3LYP/6-31+G(d) level of theory. bFrom NBO analysis at B3LYP/6-311+G(2d,p) level of theory.

calculated n → π* stabilization energy is 1.28 kcal/mol for AcPro-OMe, similar to the previously reported value,42 and 1.02 kcal/mol for Ac-Thp-OMe. The smaller n → π* stabilization energy of Ac-Thp-OMe molecule might explain why the PPII structure was destabilized upon introduction of Thp into a polyproline peptide as observed from CD measurements. To learn more insights into the impacts of Thp on a peptide structure, DFT calculations were also conducted on the conformation of oligopeptides. We used Ac-(Pro)2X(Pro)2OMe as a model system because it was found to be the simplest modified polyproline system that can form PPI and PPII conformations.44 The energy-minimized structure of Ac-(Pro)5OMe was used to construct the initial conformation of Thp-

Figure 4. Molecular structures of (A) Ac-Pro-OMe and (B) Ac-ThpOMe with trans amide bonds. These structures were generated by energy minimization using the program of Gaussian 09; the hydrogen atoms are omitted for clarity. 10817



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Table 5. Dihedral Angles and Energy Differences Calculated from the Energy-Optimized PPI Structures in Gas Phasea N → C dihedral anglesc (deg) peptide P5 P5 ThpP5 ThpP5

ring pucker


All-endo Pro3 exo All-endo

−1.06

b

−0.03

Thp exo

ϕ1

ψ1

χ1

ϕ2

ψ2

χ2

ϕ3

ψ3

χ3

ϕ4

ψ4

χ4

ϕ5

ψ5

χ5

−82 −83 −84

161 160 160

34 34 34

−79 −79 −80

162 165 167

34 35 35

−76 −67 −77

165 164 169

33 −16 39

−74 −75 −69

165 166 168

33 33 34

−77 −76 −74

172 175 176

33 33 34

−82

160

34

−80

162

34

−77

164

−14

−74

168

34

−75

179

34

Calculated at B3LYP/6-31+G(d) level of theory. P5 is Ac-(Pro)5-OMe, and Thp-P5 is Ac-(Pro)2-Thp-(Pro)2-OMe. bΔE is the minimized energy of the PPI conformation with all residues in an endo pucker relative to the respective PPI conformation with Pro3 or Thp in an exo pucker. cAn ideal PPI helix has the dihedral angles of (ϕ, ψ) = (−75°, 160°). a

Table 6. Dihedral Angles and Energy Differences Calculated from the Energy-Optimized PPI Structures in n-Propanola N → C dihedral anglesc (deg) peptide P5 P5 ThpP5 ThpP5

ring pucker


All-endo Pro3 exo All-endo

−0.82

Thp exo

−0.39

b

ϕ1

ψ1

χ1

ϕ2

ψ2

χ2

ϕ3

ψ3

χ3

ϕ4

ψ4

χ4

ϕ5

ψ5

χ5

−83 −82 −82

162 162 161

33 33 33

−79 −80 −79

162 166 163

33 34 33

−76 −67 −78

165 163 166

33 −18 39

−77 −78 −74

165 165 165

33 33 33

−79 −78 −78

172 176 179

33 33 34

−82

161

33

−80

164

33

−75

162

−17

−75

166

32

−77

180

33

Calculated at B3LYP/6-31+G(d) level of theory. P5 is Ac-(Pro)5-OMe, and Thp-P5 is Ac-(Pro)2-Thp-(Pro)2-OMe. bΔE is the minimized energy of the PPI conformation with all residues in an endo pucker relative to the respective PPI conformation with Pro3 or Thp in an exo pucker. cAn ideal PPI helix has the dihedral angles of (ϕ, ψ) = (−75°, 160°). a

substituted Ac-(Pro)2Thp(Pro)2-OMe peptide with the χ torsion angle of Thp set at 0 or 19° to monitor the conformational preference of Thp in polyproline structure. In optimized PPII conformation, torsion angles of Ac-(Pro)2Thp(Pro)2-OMe are similar to those of Ac-(Pro)5-OMe (Table S1 in the Supporting Information). We also calculated the relative energies for exo-, endo-puckered Pro and Thp in Ac(Pro)2X(Pro)2-OMe from the optimized PPII conformation in water. As shown in Table S2 (in the Supporting Information), the proline in Ac-(Pro)5-OMe readily adopted an exo pucker upon energy minimization even the initial structure was set to an endo pucker. No local minimum structures could be found for endo-puckered Pro in PPII conformation, indicating that exo-puckered Pro residues predominate in PPII structure. For Ac-(Pro)2Thp-(Pro)2OMe, endo-puckered Thp is less stable than exo-puckered Thp by a marginal energy of 0.47 kcal/mol, suggesting that two puckered forms of Thp likely coexist in the peptide affecting the stability of PPII conformation. The calculated results of Ac(Pro)2X(Pro)2-OMe in water are consistent with the fact that the Thp-containing peptides form a less stable PPII helix (Figure 1 and Table 2) and Thp has a weaker backbone n → π* interaction (Table 4). Likewise, the energy-minimized PPI structures for the Ac-(Pro)2X(Pro)2-OMe peptide were obtained and torsion angles of Ac-(Pro)2Thp(Pro)2-OMe in optimized PPI conformation differ only slightly from those of Ac-(Pro)5-OMe (Table 5). However, during the process of geometry optimization for the PPI conformation of Ac(Pro)2Thp(Pro)2-OMe, the ring pucker of Thp was found to change readily from the initial endo (χ = 19°) pucker to an exo (χ = −14°) pucker. Thereby, we calculated the energy difference between the PPI conformations of Ac-(Pro)2Thp(Pro)2-OMe with all-endo puckers and with Thp exo puckered and found that the difference is only 0.03 kcal/mol (Table 5).

In a similar manner, the energy difference between the PPI conformations of Ac-(Pro)5-OMe with all-endo puckers and with Pro3 exo puckered was calculated to be 1.03 kcal/mol. The calculations were further conducted in the presence of npropanol to examine the energy difference in solution. As shown in Table 6 the dihedral angles of P5 and Thp-P5 in optimized PPI conformation are similar to those calculated in gas phase, and the energy difference between endo and exo puckers for Thp-P5 is smaller than that for P5 (0.39 vs 0.82 kcal/mol), concurring with the results in gas phase. These results strongly suggest that Thp can interconvert between endo and exo puckers more readily than Pro, even in a PPI helix. Because the exo pucker is not favored in a PPI helix, the coexistence of endo and exo puckers in Thp residues may lead to a low content of PPI conformation in solution. In addition, the Gibbs free energy difference between allendo puckers and with the middle residue exo puckerd was also calculated in n-propanol. As shown in Table 7, P5 strongly favors an all-endo puckered structure over the Pro3-exo puckered structure by 2.04 kcal/mol in a PPI helix while the Thp-endo pucker structure is slightly more stable than the Thpexo pucker in Thp-P5 by 0.66 kcal/mol, indicating that Thp-P5 form a less stable PPI helix. In addition, the activation energy (Ea) of interconverting between endo and exo puckers for the peptide models was also calculated in solution. In n-propanol, the activation energy of converting an endo pucker to an exo pucker is 1.50 kcal/mol and that of converting an exo to an endo pucker is 0.83 kcal/mol for Thp-P5, which is smaller than the corresponding activation energy for P5 (3.44 and 1.39 kcal/ mol), suggesting that the pucker of Thp can readily interconvert between endo and exo conformations and affect the PPI stability of Thp-P5. The computational results in solution are consistent with the CD spectrum of Thp-P11 in npropanol (Figure 1B) and its low thermal stability in PPI 10818



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energy-optimized PPII structures, and the calculated relative energies for exo- and endo-puckered Ac-(Pro)2X(Pro)2-OMe with PPII conformation in water are included. This material is available free of charge via the Internet at http://pubs.acs.org.

Table 7. Thermodynamic Parameters and Activation Energy Calculated from the Energy-Optimized PPI Structures in nPropanola peptide P5 P5 ThpP5 ThpP5

ring pucker Allendo Pro3 exo Allendo Thp exo

Δ(Hendo− Hexo)b (kcal/mol)

Δ(Sendo− Sexo)b (cal mol−1 K−1)

Δ(Gendo− Gexo)b (kcal/mol)

Eac (kcal/mol)

−0.89

3.89

−2.04

3.44

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*E-mail: [email protected]. Address: Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 30013, R.O.C.

1.39 −0.36

1.03

−0.66

1.50

Notes

0.83

The authors declare no competing financial interest.

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a

Calculated at B3LYP/6-31+G(d) level of theory. P5 is Ac-(Pro)5OMe, and Thp-P5 is Ac-(Pro)2-Thp-(Pro)2-OMe. bΔH, ΔS, and ΔG are the differences between the PPI conformation with all residues in an endo pucker and the respective PPI conformation with Pro3 or Thp in an exo pucker. cEa is the activation energy, calculated from the energy difference between the transition state and the ground state.

ACKNOWLEDGMENTS This work was supported by Taiwan Ministry of Science and Technology (Grants NSC 101-2113-M-007-017-MY2 and NSC 98-2119-M-007-011) and National Tsing Hua University (Grants 102N2011E1 and 103N2011E1). We are grateful to the National Center for High-performance Computing (NCHC) for computer time and facilities. We also thank Mr. Goa-Tao Huang and Mr. Pei-Kang Tsou for their assistance on DFT calculations.

structure (Figure 2). Unlike the other 4-substituted proline derivatives, for example, (2S,4S)-4-fluoroproline and (2S,4S)-4methoxyproline,43 the low Ktrans/cis of Thp does not reflect on the propensity to form PPI helices, suggesting that the larger sulfur atom and ring side chain may complicate the interactions; as a result, Thp may exhibit different structural features in a polypeptide. One of the perspectives could be that using some other techniques, such as Raman optical activity (ROA), to study the polyproline peptides with Thp or other proline derivatives incorporated to complement CD measurements.45 Moreover, our computational results indicate that such analyses may be extended to investigate the impacts of other proline derivatives and analogs on polyproline conformation, which can be helpful to interpret the experimental observations on these unique structures.

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REFERENCES

(1) Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T. Conformational Stability of Collagen Relies on a Stereoelectronic Effect. J. Am. Chem. Soc. 2001, 123, 777−778. (2) Pal, D.; Chakrabarti, P. Cis peptide bonds in proteins: residues involved, their conformations, interactions and locations. J. Mol. Biol. 1999, 294, 271−288. (3) Milner-White, E. J.; Bell, L. H.; Maccallum, P. H. Pyrrolidine ring puckering in cis and trans-proline residues in proteins and polypeptides: Different puckers are favoured in certain situations. J. Mol. Biol. 1992, 228, 725−734. (4) DeTar, D. F.; Luthra, N. P. Conformations of Proline. J. Am. Chem. Soc. 1977, 99, 1232−1244. (5) Madison, V. Flexibility of the pyrrolidine ring in proline peptides. Biopolymers 1977, 16, 2671−2692. (6) Vitagliano, L.; Berisio, R.; Mastrangelo, A.; Mazzarella, L.; Zagari, A. Preferred proline puckerings in cis and trans peptide groups: Implications for collagen stability. Protein Sci. 2001, 10, 2627−2632. (7) Balasubramanian, R.; Lakshminarayanan, A. V.; Sabesan, M. N.; Tegoni, G.; Venkatesan, K.; Ramachandran, G. N. Studies on the conformation of amino acids. Int. J. Protein Res. 1971, 3, 25−33. (8) 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. (9) Hinderaker, M. P.; Raines, R. T. An electronic effect on protein structure. Protein Sci. 2003, 12, 1188−1194. (10) Horng, J.-C.; Raines, R. T. Stereoelectronic effects on polyproline conformation. Protein Sci. 2006, 15, 74−83. (11) Kümin, M.; Sonntag, L. S.; Wennemers, H. Azidoproline Containing Helices: Stabilization of the Polyproline II Structure by a Functionalizable Group. J. Am. Chem. Soc. 2007, 129, 466−467. (12) Sonntag, L.-S.; Schweizer, S.; Ochsenfeld, C.; Wennemers, H. The “Azido Gauche Effect”-Implications for the Conformation of Azidoprolines. J. Am. Chem. Soc. 2006, 128, 14697−14703. (13) Crespo, M. D.; Rubini, M. Rational design of protein stability: Effect of (2S,4R)-4-fluoroproline on the stability and folding pathway of ubiquitin. PLoS One 2011, 6, e19425. (14) Moroder, L.; Budisa, N. Synthetic biology of protein folding. ChemPhysChem 2010, 11, 1181−1187. (15) Naduthambi, D.; Zondlo, N. J. Stereoelectronic Tuning of the Structure and Stability of the Trp Cage Miniprotein. J. Am. Chem. Soc. 2006, 128, 12430−12431.

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CONCLUSIONS In this work, we report the consequences of substituting proline with thiaproline on polyproline conformation. Although the ring geometry of Thp is relatively similar to that of Pro, Thp shows a dramatically different tendency to form PPI and PPII helices. For Thp, its preference for an endo ring pucker and a cis peptide bond results in a destabilized PPII conformation, as expected and rationalized by its weaker backbone n → π* interactions. Strikingly, such a preference does not make Thp favor a PPI structure. Instead, Thp-containing peptides form unstable PPI conformation, and increasing the number of Thp residues can eventually prohibit the formation of PPI helices. By combining experimental data and computational analysis, we have demonstrated that the low energy barrier between the endo and exo ring puckers of Thp could be the key factor in affecting PPI conformation. The readiness of ring pucker interconversion makes the thiazolidine ring of Thp highly flexible, leading to perturbation of a typical PPI helix. In conclusion, our study extends the understanding of polyproline conformation with Thp being incorporated, and the findings reveal new important information to design Thp-containing peptides as biomedical scaffolds or templates.

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S Supporting Information *

First derivatives of thermal unfolding curves, CD spectra before and after being heated, the dihedral angles calculated from the 10819



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(16) Rubini, M.; Scharer, M. A.; Capitani, G.; Glockshuber, R. (4R)and (4S)-Fluoroproline in the conserved cis-prolyl peptide bond of the thioredoxin fold: tertiary structure context dictates ring puckering. ChemBioChem 2013, 14, 1053−1057. (17) Shoulders, M. D.; Raines, R. T. Collagen structure and stability. Annu. Rev. Biochem. 2009, 78, 929−958. (18) Tang, H.-C.; Lin, Y.-J.; Horng, J.-C. Modulating the folding stability and ligand binding affinity of Pin1 WW domain by proline ring puckering. Proteins 2014, 82, 67−76. (19) 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. (20) Borgogno, A.; Ruzza, P. The impact of either 4-R-hydroxyproline or 4-R-fluoroproline on the conformation and SH3m‑cort binding of HPK1 proline-rich peptide. Amino Acids 2013, 44, 607−614. (21) Holzberger, B.; Obeid, S.; Welte, W.; Diederichs, K.; Marx, A. Structural insights into the potential of 4-fluoroproline to modulate biophysical properties of proteins. Chem. Sci. 2012, 3, 2924−2931. (22) Kim, W.; McMillan, R. A.; Snyder, J. P.; Conticello, V. P. A Stereoelectronic Effect on Turn Formation Due to Proline Substitution in Elastin-Mimetic Polypeptides. J. Am. Chem. Soc. 2005, 127, 18121−18132. (23) Goodman, M.; Chen, V.; Benedetti, E.; Pedone, C.; Corradini, P. Conformational aspects of polypeptide structure. XLI. Cystal structure of S-thiazolidine-4-carboxylic acid and helical structure of poly[(S)-thiazolidine-4-carboxylic acid]. Biopolymers 1972, 11, 1779− 1787. (24) Budisa, N.; Minks, C.; Medrano, F. J.; Lutz, J.; Huber, R.; Moroder, L. Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: Structure and stability of the per-thiaproline mutant of annexin V. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 455−459. (25) Benedetti, E.; Christensen, A.; Gilon, C.; Fuller, W.; Goodman, M. Conformational studies of peptides. The crystal structures of Nacetyl-L-prolinamide and N-acetyl-(S)-thiazolidine-4-carboxamide. Biopolymers 1976, 15, 2523−2534. (26) Kang, Y. K. Cis−Trans Isomerization and Puckering of Pseudoproline Dipeptides. J. Phys. Chem. B 2002, 106, 2074−2082. (27) Robert, F. The crystal and molecular structure of N-tertbutyloxycarbonyl-l-thiazolidine-4-carboxylic acid (C9H15NO4S). Acta Crystallogr., Sect. B 1976, 32, 2367−2369. (28) 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. (29) Shuman, R. T.; Rothenberger, R. B.; Campbell, C. S.; Smith, G. F.; Gifford-Moore, D. S.; Paschal, J. W.; Gesellchen, P. D. StructureActivity Study of Tripeptide Thrombin Inhibitors Using α-Alkyl Amino Acids and Other Conformationally Constrained Amino Acid Substitutions. J. Med. Chem. 1995, 38, 4446−4453. (30) Kern, D.; Schutkowski, M.; Drakenberg, T. Rotational Barriers of cis/trans Isomerization of Proline Analogues and Their Catalysis by Cyclophilin. J. Am. Chem. Soc. 1997, 119, 8403−8408. (31) Gorres, K. L.; Edupuganti, R.; Krow, G. R.; Raines, R. T. Conformational Preferences of Substrates for Human Prolyl 4Hydroxylase. Biochemistry 2008, 47, 9447−9455. (32) Lubec, B.; Hjelm, M.; Hoeger, H.; Gialamas, J.; Lubec, G. 4thiaproline reduces heart lipid peroxidation and collagen accumulation in the diabetic db/db mouse. Amino Acids 1997, 12, 343−351. (33) Prabhakar, Y.; Raja Solomon, V.; Gupta, M.; Katti, S. B. QSAR studies on thiazolidines: A biologically privileged scaffold. In QSAR and Molecular Modeling Studies in Heterocyclic Drugs II; Gupta, S., Ed.; Springer: Berlin Heidelberg, 2006; Vol. 4, pp 161−249. (34) Witter, D. J.; Famiglietti, S. J.; Cambier, J. C.; Castelhano, A. L. Design and synthesis of SH3 domain binding ligands: Modifications of the consensus sequence XPpXP. Bioorg. Med. Chem. Lett. 1998, 8, 3137−3142.

(35) Choudhary, A.; Pua, K.; Raines, R. Quantum mechanical origin of the conformational preferences of 4-thiaproline and its S-oxides. Amino Acids 2011, 41, 181−186. (36) Kang, Y. K.; Park, H. S.; Byun, B. J. Puckering transitions of pseudoproline residues. Biopolymers 2009, 91, 444−455. (37) Kang, Y. K.; Park, H. S. Conformational Preferences of Pseudoproline Residues. J. Phys. Chem. B 2007, 111, 12551−12562. (38) Kuemin, M.; Nagel, Y. A.; Schweizer, S.; Monnard, F. W.; Ochsenfeld, C.; Wennemers, H. Tuning the cis/trans conformer ratio of Xaa-Pro amide bonds by intramolecular hydrogen bonds: The effect on PPII helix stability. Angew. Chem., Int. Ed. 2010, 49, 6324−6327. (39) Brunck, T. K.; Weinhold, F. Quantum-Mechanical Studies on the Origin of Barriers to Internal Rotation about Single Bonds. J. Am. Chem. Soc. 1979, 101, 1700−1709. (40) Goodman, M.; Niu, G. C. C.; Su, K.-C. Conformational Aspects of Polypeptide Structure. XXXI. Helical poly[(S)-thiazolidine-4carboxylic acid] and poly[(S)-oxazolidine-4-carboxylic acid]. Theoretical Results. J. Am. Chem. Soc. 1970, 92, 5219−5220. (41) Goodman, M.; Su, K.-C.; Niu, G. C. C. Conformational Aspects of Polypeptide Structure. XXXII. Helical poly[(S)-thiazolidine-4carboxylic acid]. Experimental Results. J. Am. Chem. Soc. 1970, 92, 5220−5222. (42) DeRider, M. L.; Wilkens, S. J.; Waddell, M. J.; Bretscher, L. E.; Weinhold, F.; Raines, R. T.; Markley, J. L. Collagen Stability: Insights from NMR Spectroscopic and Hybrid Density Functional Computational Investigations of the Effect of Electronegative Substituents on Prolyl Ring Conformations. J. Am. Chem. Soc. 2002, 124, 2497−2505. (43) Wendt, W.; Weinhold, F. NBOView 1.1., Theoretical Chemistry Institute; University of Wisconsin-Madison: Madison WI, 2001. (44) Zhong, H.; Carlson, H. A. Conformational studies of polyprolines. J. Chem. Theory Comput. 2006, 2, 342−353. (45) Profant, V.; Baumruk, V.; Li, X.; Šafařík, M.; Bouř, P. Tracking of the Polyproline Folding by Density Functional Computations and Raman Optical Activity Spectra. J. Phys. Chem. B 2011, 115, 15079− 15089.

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The Impact of 4-Thiaproline on Polyproline Conformation - The

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