Cation−π Interaction Induced Folding of AAB-Type Collagen

Jan 28, 2016 - Collagen is the most predominant component of the extracellular matrix. Natural collagens consist of all identical (AAA, homotrimer), t...
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Cation−π Interaction Induced Folding of AAB-Type Collagen Heterotrimers Chu-Harn Chiang† and Jia-Cherng Horng*,†,‡ †

Department of Chemistry, National Tsing Hua University, 101 Sec. 2 Kuang-Fu Road, Hsinchu, Taiwan 30013, R.O.C. Frontier Research Center on Fundamental and Applied Science of Matters, National Tsing Hua University, 101 Sec. 2 Kuang-Fu Road, Hsinchu, Taiwan 30013, R.O.C.

‡

S Supporting Information *

ABSTRACT: Collagen is the most predominant component of the extracellular matrix. Natural collagens consist of all identical (AAA, homotrimer), two different (AAB, heterotrimer), or three different (ABC, heterotrimer) peptide chains. Many natural collagens are either AAB- or ABC-type heterotrimers, making heterotrimeric helices better mimics for studying collagen structures in nature. We prepared collagen-mimetic peptides containing cationic (Arg) or aromatic (Phe, Tyr) residues to explore collagen heterotrimer folding via cation−π interactions. Circular dichroism, differential scanning calorimetry, and nuclear magnetic resonance (NMR) measurements showed that the interchain cation−π interactions between cationic and aromatic peptides could induce AAB-type heterotrimer formation. By controlling the mixing molar ratios of cationic and aromatic peptides in solution, we could obtain the heterotrimers with various compositions. We demonstrate the effectiveness of cation−π interactions as a force to fold collagen heterotrimers.

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INTRODUCTION Collagen is the most abundant protein in the human body, making up 25−35% of the total-body protein content.1 It is usually found in fibrous and connective tissues such as tendons, skin, bones, and blood vessels. Collagen is a right-handed triple helix consisting of three left-handed polyproline II (PPII) helices, and is normally composed of Xaa-Yaa-Gly triplet amino acid repeats with one amino acid offset between the peptide chains. The one-residue offset maximizes the interpeptide hydrogen bond and optimizes triple helix packing. Glycine without a bulky side chain is required every three residues to avoid steric clashes within the triple helix. The Xaa and Yaa positions of the repeated sequences are often proline (Pro, P) and hydroxyproline (Hyp, O). On the basis of the differences in peptide chains, 28 types of fibrillar collagen have currently been identified, among which most types are composed of heterotrimeric helices. In particular, type I collagen, which constitutes at least 90% of the total collagen in the human body, is an AAB-type heterotrimer (two α1 chains and one α2 chain). The disorders and mutations in the structure of this type are associated with the diseases such as osteogenesis imperfect and Ehlers−Danlos syndrome.2−5 Owing to its large size, insolubility, repetitive sequence, and complex hierarchical structure, it was relatively difficult to perform detailed biochemical and biophysical analyses on native collagen. Thus, collagen-mimetic peptides (CMPs) have been introduced to the study of collagen since the late 1960s. Most work has used homotrimers as mimics to examine the assembly and folding of collagen.6 Although the previous studies using homotrimetric models revealed many insights into native collagen structure,7,8 a more nature-mimetic system is desired for collagen study. To probe the diseases that are © XXXX American Chemical Society

related to the dysfunction and structural faults of type I collagen, it would be more appropriate to use AAB-type heterotrimeric helices as the mimics to study the folding of this class of nature collagen. In the past, the strategies employed to prepare heterotrimers mainly used covalent bonds such as disulfide bonds or cysteine knots,9−12 branched linkers,6 and template-tethered approaches.13 Recent work also showed that collagen heterotrimers could be formed via metal−peptide coordination14 or mixing the fragments with natural type IV collagen sequences.15 The NC2 domain of type IX collagen was also shown to be useful in building stagger-specific heterotrimers.15 Hartgerink and co-workers first used electrostatic interactions to successfully fold a collagen heterotrimer.16,17 Subsequent work found that such interactions could effectively induce the formation of various heterotrimeric helices and control their self-assembly into large constructs.18−26 In addition to electrostatic interactions, the cation−π interaction, which is a noncovalent force and regarded as an electrostatic attraction between a positive charge and the quadrupole moment of the aromatic ring, is also frequently observed in proteins.27 This interaction has been found to be important for protein structures and functions and has a wide variety of applications in biology and materials.28,29 Previously, we used a series of peptide models to show that the introduction of cation−π interactions can stabilize the collagen triple helices30 and very effectively promote the selfassembly of small CMPs into fibrils.31 On the basis of that work, we attempted to extend the application of cation−π Received: November 15, 2015 Revised: January 23, 2016

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DOI: 10.1021/acs.jpcb.5b11189 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

concentrations of peptides were 0.2 mM for all the measurements. Thermal unfolding curves were obtained by monitoring the ellipticity at 225 nm. For thermal unfolding experiments, the data were recorded every 1 °C with an average heating rate of 0.16 °C/min (1 °C/min heating and 5 min equilibrium). Values of Tm were determined by taking first derivatives of the melting curves versus temperature. Refolding Kinetics. For kinetic CD experiments, peptide samples were prepared in the same manner as that in the thermal denaturation experiments. The samples were heated at 80 °C for 20 min, and then immediately transferred to a 1 mm cuvette that had been precooled at 4 °C. The data were collected for 6 h. Refolding half-time (t1/2), the time required to reach the fraction folded (F) of 0.5, was used as an index of refolding rate. The fraction folded was calculated using the following equation

interactions to the folding of collagen heterotrimers. In the present study, we designed and prepared three collagenmimetic peptides to explore whether cation−π interactions could induce the folding of a heterotrimeric helix. By combining circular dichroism (CD), differential scanning calorimerty (DSC), and nuclear magnetic resonance (NMR) measurements, we showed that the installation of interchain cation−π interactions could effectively induce the folding of an AAB-type collagen heterotrimer.

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MATERIALS AND METHODS General. Chemical reagents and Fmoc-protected amino acids were purchased form Advanced Chemtech, Alfa Aesar, ECHO, J.T. Baker, Sigma-Aldrich, and used without further purification. 15N-isotopically enriched Gly was purchased from Sigma-Aldrich. Fmoc-Pro-Hyp-Gly-OH tripeptide was synthesized in solution as described previously.32 Peptide Synthesis and Purification. The peptides used in this study are listed in Table S1 of the Supporting Information. All peptides were synthesized on a 0.05 mmol scale by solid phase synthesis using the Fmoc-Pro-Hyp-Gly-OH tripeptide, Fmoc-protected amino acids, HBTU-mediated coupling, and standard reaction cycles on a PS3 synthesizer (Protein Technologies). Use of a Rink amide resin with a MBHA linker generated an amidated C-terminus following the cleavage. The N-terminus of each peptide was acetylated using acetic anhydride upon the completion of synthesis. Cleavage of the peptides from resins and removal of side-chain protecting groups were carried out with a solution of 95% trifluoroacetic acid (TFA)/2.5% triisopropylsilane (TIS)/2.5% H2O (v/v) or 94% TFA/1% TIS/2.5% H2O/2.5% ethandithiol (v/v). Reverse phase HPLC with a YMC semipreparative C18 column was used to purify the peptides. H2O/acetonitrile gradients with 0.1% (w/v) TFA as the counterion were used to elute the peptides. Purity of the peptides was more than 90% according to HPLC analysis (Figure S1 of the Supporting Information). A MALDI-TOF mass spectrometer (Autoflex III Smartbeam LRF-200-CID, Bruker Daltonics) was used to confirm the identity of the peptides, and their measured masses are shown in Table S1 and Figure S2 of the Supporting Information. For 15 N-labeled peptides, the middle Gly residue of each peptide was substituted by an 15N-labeled Gly residue, and their syntheses were carried out with standard solid phase methods as previously mentioned. Circular Dichroism (CD) Spectroscopy. CD measurements were performed using an Aviv model 410 CD spectrometer with 1 mm path length quartz cuvette. For homotrimer experiments, prior to measurements the peptides were dissolved in pH 7.0 and 20 mM sodium phosphate buffer and heated to 80 °C to denature the triple helices, and then incubated at 4 °C for at least 24 h to allow the formation of triple helices. Although aromatic peptides (F and Y) were not very soluble in buffered solution at room temperature, they could completely dissolve in solution at 80 °C, and the aromatic peptide solutions remained clear without any precipitates before and after the measurements. For heterotrimer experiments, the samples containing cationic (R) and aromatic (F or Y) peptides with different molar ratios were first prepared in pH 7.0 and 20 mM sodium phosphate buffer and then heated at 80 °C for 20 min. After the heating, the solutions were cooled down to room temperature for 15 min and then incubated at 4 °C for at least 24 h before CD measurements to allow the self-assembly of CMPs. The total

F=

θt − θu θf − θu

(1)

where θt is the ellipticity at time t and θf is the ellipticity of the folded form at 4 °C, which was obtained by measuring the same sample after 24 h incubation and used as the fully formed baseline; θu is the ellipticity of the unfolded form at 4 °C, which was determined by fitting the refolding curve to time zero. Differential Scanning Calorimetry (DSC) Analysis. DSC curves were acquired using a MicroCal VP-DSC instrument at National Chung Hsing University Instrument Center and processed using the affiliated Origin software. Before DSC measurements, the samples were prepared in a same manner as that in the CD measurements with a total peptide concentration of 0.6 mM. The peptide solutions were degassed at 15 °C for more than 5 min prior to measurements. Samples were scanned from 15 to 80 °C with a heating rate of 0.1 °C/ min. A progress baseline was subtracted from each DSC curve before analysis. The Tm value was obtained at the maximum of each transition in the DSC endotherm. Values of ΔH (per mole of monomer) were obtained by direct integration of the DSC endotherm for the samples containing one major component. The value of ΔS at the Tm was calculated by the following equation Tm =

ΔH [ΔS + R ln(0.75c 2)]

(2)

where c is the concentration of monomeric peptide and R is the universal gas constant. In our analysis, we assumed that ΔCp is 0 for triple-helix unfolding, and ΔH and ΔS are independent of temperature. Values of ΔG were calculated as ΔG = ΔH − TΔS. Nuclear Magnetic Resonance (NMR) Spectroscopy. NMR experiments were performed on a Bruker DMX 600 MHz spectrometer at the National Tsing Hua University Instrumentation Center. 1H,15N-HSQC (heternuclear single quantum coherence), TOCSY (total correlation spectroscopy), and NOESY (nuclear Overhauser effect spectroscopy) spectra were taken with a peptide concentration of 2 mM in 90% H2O/ 10% D2O, pH 7.0, and 20 mM phosphate buffer. A mixing time of 75 ms was used in the TOCSY experiments, and 400 ms was used in the NOESY experiments. The TOCSY and NOESY spectra were taken at 10 °C and internally referenced to sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) at 0.0 ppm. For the heterotrimer samples, the peptide solutions were first heated at 80 °C for 20 min, and were then cooled down at B

DOI: 10.1021/acs.jpcb.5b11189 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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After the preparation of peptides, we used CD spectroscopy to characterize their structures. Before CD measurements, all the peptide solutions were heated to 80 °C to denature the triple helices, and then incubated at 4 °C for more than 24 h to allow refolding of the triple helices. As shown in Figure 2, CD

room temperature for 15 min followed by the incubation at 4 °C for at least 24 h before taking NMR spectra. The 1H,15NHSQC experiments were conducted at a low temperature (10 °C) and a high temperature (40−50 °C) to display the resonances for monomers and trimers as the triple helices would dissociate into the monomeric forms at high temperatures. The 1H,15N-HSQC spectra of R, F, and Y peptides were also recorded as shown in Figures S6 and S7 of the Supporting Information for comparison. Molecular Modeling. The structure (PDB ID: 2KLW)33 from the Protein Data Bank was used as a basis for molecular modeling. The original sequence in 2KLW was modified into our sequence for structural optimization. The energy minimization was performed using the Discovery Studio 4.0 software with the CHARMm force field (version c37b2) applied. The calculation program and facility were provided by the National Center for High-Performance Computing, Taiwan. The initial structure was generated by energy minimization while locking the peptide backbone with harmonic restraints. The Adopted Basis NR algorithm was used, and the force constant was conducted with 20, 10, 5 kcal/ mol and 15 000, 5000, 5000 steps. Following the minimization, the backbone was unconstrained and calculated with a force constant of 5 kcal/mol and 5000 steps to give the final energyminimized structure. From the optimized structure, the cation−π pair within 6 Å was assigned.

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RESULTS AND DISCUSSION Our previous study on collagen homotrimers indicated that a favorable cation−π interaction could be generated to stabilize the triple helix if the cationic residues were at position Yaa, and the aromatic residues were at position Xaa in the sequence. However, no cation−π stabilization could occur if the relative positions were reversed.30 Accordingly, we designed an R peptide with Arg incorporated into the Yaa position and the F and Y peptides with Phe or Tyr implanted at position Xaa. Due to the low solubility of aromatic CMPs in water, the designed peptides only contain either four cationic or aromatic residues, as shown in Figure 1A. Moreover, all the peptides have an acetylated N-terminus and an amidated C-terminus to avoid the electrostatic repulsions between individual peptides. From this design, we inferred that the cationic peptide (R) could associate with the aromatic peptide (F or Y) to form collagen heterotrimers via interchain cation−π interactions once they were mixed in solution (Figure 1B,C).

Figure 2. (A) Thermal unfolding curves monitored by CD for R, F, and the mixtures of R and F. (B) The first derivatives of the thermal unfolding curves versus temperature. All the measurements were conducted in pH 7.0 and 20 mM phosphate buffer using a peptide concentration of 0.2 mM and an average heating rate of 0.16 °C/min.

measurements indicate that R can form homotrimers (R3) with a Tm value of 49 °C while F cannot fold into triple helices. All the mixtures of R and F also exhibit a collagen characteristic thermal unfolding transition and have a lower Tm value than that of R3 (Table 1), suggesting the formation of collagen heterotrimers in solution. Another piece of evidence showing the formation of R-F heterotrimeric constructs was given by the observation that the F peptide solution was cloudy when the peptides were initially dissolved in the pH 7.0 buffer at room temperature because F is not very soluble in water, but the solution became transparent upon mixing with R peptide. More interestingly, two Tm values could be obtained as the mixing molar ratios of F to R increased. The two Tm values (35 and 47 °C) are identical for the mixtures of 1R:1F and 1R:2F, and the higher one is the same as the Tm value of 2R:1F mixture,

Figure 1. (A) Sequences of the peptides R, F, and Y, where Hyp is 4Rhydroxyproline. All the peptides have an acetylated N-terminus and an amidated C-terminus. (B) Illustration of the interchain cation−π cation in an R-F heterotrimer. (C) The relative position of Phe, Arg, and Gly viewed along the axis from the N-terminus. The diagrams were generated using the PDB entry 1CAG.30 C

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The Journal of Physical Chemistry B Table 1. Tm Values Measured by CD and DSC for the Peptide Mixtures and the Major Compositions in Solution at pH 7.0 peptide mixture R only F only Y only 2R:1F 1R:1F 1R:2F 2R:1Y 1R:1Y 1R:2Y

Tm (°C) by CDa Tm (°C) by DSCc 49 no triple helix no triple helix 47 35, 47b 35, 47 47 35, 47 34, 47

48.7 no triple helix no triple helix 45.3 36.4, 45.4 35.0, 45.3 45.9 34.0, 45.8 34.0, 45.8

To further examine the folding and thermal stability of heterotrimers, we conducted DSC analysis on the R-only solution and R-F mixtures. As shown in Figure 3, the R3 homotrimer exhibits one transition peak with a Tm value of 48.7 °C, and the 2R:1F mixture also has only one transition peak with a Tm value of 45.3 °C, consistent with the CD measurements. Only one transition was found in both CD measurements and DSC analysis for the 2R:1F mixture, strongly suggesting that an AAB-type of heterotrimer (R2F1) predominates in solution. Both the 1R:1F and 1R:2F mixtures display two transitions peaks in DSC profiles, which give two almost identical sets of Tm values, 36.4 and 45.4, and 35.0 and 45.3 °C, respectively. From DSC analysis, it is evident that two types of heterotrimers exist in the 1R:1F and 1R:2F mixtures. We speculated that the transition peak at 45.3 °C represents the R2F1 heterotrimer while that at 36.4 °C represents the R1F2 heterotrimer. R1F2 is less stable than R2F1 because F cannot form triple helices and the heterotrimer with a high content of F will have a low stability. Furthermore, the transition of R1F2 becomes more pronounced in DSC endotherms when the molar ratio of F increases because a high concentration of F will increase the population of R1F2 in solution. This is also consistent with the CD results that increasing the molar ratio of F in the mixture would produce a more distinct transition in the lower temperature region that corresponds to the R1F2 triple helix. Similar results were also observed in the Y peptide and the mixtures of R and Y (Figures S4 and S5 of the Supporting Information and Table 1), indicating that F and Y exhibit a similar behavior and interact with R in the same manner.

major compositiond R3 F monomers Y monomers R2F1 R1F2, R2F1 R1F2, R2F1 R2Y1 R1Y2, R2Y1 R1Y2, R2Y1

The Tm values were determined by the first derivatives of the transition curves versus temperature. bThe minimum at 35 °C was not clear. cTwo values indicate that there are two transition peaks resolved in DSC endotherms. dThe major compositions and their corresponding Tm values were assigned according to CD and DSC data. a

suggesting that two types of R-F heterotrimers may form in solution. Additionally, we also performed thermal unfolding measurements on the 2R:1F mixture under different ionic strength. As shown in Figure S3 of the Supporting Information, we found that the Tm value for the sample containing 150 mM NaCl is identical to that for the sample without NaCl. The results suggest that cation−π interaction induced collagen heterotrimers are not sensitive to ionic strength, providing a useful property for this method in preparing heterotrimeric helices.

Figure 3. DSC profiles for (A) R, (B) the 2R:1F mixture, (C) the 1R:1F mixture, and (D) the 1R:2F mixture. All the measurements were conducted in pH 7.0 and 20 mM phosphate buffer using a peptide concentration of 0.6 mM and a heating rate of 6 °C/h. D

DOI: 10.1021/acs.jpcb.5b11189 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B We also prepared 15N-labeled CMPs in which the middle Gly in the sequence was substituted with 15N-labled Gly and performed NMR measurements to further confirm the formation of AAB-type collagen heterotrimers. As shown in Figure 4, the overlapped 1H,15N-HSQC spectra of R peptide

with that of R3. The refolding rates of R2F1 and R2Y1 are both faster than that of R3 (Table 2 and Figure 5), suggesting that

Figure 4. Overlapped 1H,15N-HSQC spectra of R peptide solution (blue) and the 2R:1F mixture (green) at 10 °C. The t denotes trimers while m denotes monomers.

Figure 5. Refolding kinetic measurements for the triple helices of R3, R2F1, and R2Y1 at 4 °C. All the peptides were incubated at 80 °C for 20 min prior to measurements. All the measurements were conducted in pH 7.0 and 20 mM phosphate buffer with a peptide concentration of 0.2 mM.

and the 2R:1F mixture indicate that a new cross peak different from R3 was found for the mixture. This peak suggests the formation of a heterotrimer that was assigned as the resonance of R2F1. From the overlapped 1H,15N-HSQC spectra of the 2R:1F and the 1R:2F mixtures (Figure S8 of the Supporting Information), we observed a new cross peak different from that of R2F1 and assigned it as the resonance of R1F2. The same NMR results were also found for the R-Y mixtures (Figure S9 of the Supporting Information). Our NMR data support the results of DSC analyses, indicating that only one type of heterotrimer forms in the 2R:1F or 2R:1Y mixture while two types of heterotrimers coexist in the 1R:2F or 1R:2Y mixture. Furthermore, we referred to a previous report using NMR to identify cation−π interactions in model α-helical peptides34 and conducted TOCSY and NOESY experiments on the 2R:1Y mixture to examine the interactions between Arg and Tyr within a heterotrimer. As shown in Figure S10 of the Supporting Information, the NOE cross peaks between the side chain of Arg and the aromatic ring of Tyr could be identified, indicating that there are interactions between the side chains of these residues. The data provides another piece of strong evidence that cation−π interactions take place between cationic and aromatic residues within the heterotrimeric helix. In the 2R:1F and 2R:1Y mixtures, only one AAB-type heterotrimer (R2F1 or R2Y1) predominates in the solution, allowing for further thermodynamic and kinetic analyses on their folding. To gain more insight into the folding of R2F1 and R2Y1, we measured their refolding rates and compared them

the favorable interchain cation−π interactions can accelerate the folding of an AAB-type heterotrimer. In addition, we calculated the folding free energy (ΔG) of R3, R2F1, and R2Y1 using the values of enthalpy (ΔH) and entropy (ΔS) obtained by DSC measurements. Since the thermodynamic parameters are most accurate near the Tm,35 the ΔG and TΔS values were calculated at 47 °C, which is the average of the Tm values of R3, R2F1, and R2Y1 measured by DSC. As shown in Table 2, all three of the triple helices have a similar stability. The folding of R2F1 and R2Y1 induces a more unfavorable ΔS value than that of R3, indicating that the folding of these two heterotrimers may need to pay a greater entropic penalty. In contrast, the formation of R2F1 and R2Y1 generates a more favorable ΔH value (more exothermic) than that of R3, strongly suggesting that the interchain cation−π interactions are the key contributor to this exothermic consequence during the folding. The thermodynamic results also show that the folding of R2F1 and R2Y1 is an enthalpy-driven process. Analysis of the thermodynamic and kinetic studies showed that the folds of R2F1 and R2Y1 are slightly less stable than that of R3, but they fold at a much faster rate than R3. This demonstrates that the formation of R2F1 or R2Y1 is favored over that of the R3 homotrimer in the mixtures and could result from kinetic control. Moreover, the refolding rate observed for either the mixture of R2F1 and R1F2 or the mixture of R2Y1 and R1Y2 is also more rapid than that of R3 (Table S2 and Figure S11 of the Supporting Information), again indicating that the introduction

Table 2. Thermodynamic Data Derived from DSC Analysis and Refolding Rates Monitored by CD Spectroscopy for Collagen Triple Helices at pH 7.0 triple helix

Tm (°C)

ΔH (kJ/mol)

−TΔS (kJ/mol)

ΔGa (kJ/mol)

t1/2b (min)

R3 R2F1 R2Y1

48.7 45.3 45.9

−49.5 −61.8 −55.8

8.94 21.8 15.8

−40.5 −39.9 −40.1

>500 193 120

TΔS and ΔG were calculated at 47 °C, the averaged Tm values of these trimers. bt1/2 is the time required for the fraction folded to reach 50%, used for folding rate comparison. The values were obtained at 4 °C.

a

E

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of the cation−π interaction favors AAB-type collagen heterotrimer folding. Furthermore, the experimental data also suggest that the formation of R2F1 and R2Y1 is more favorable than that of R1F2 and R1Y2. In the triple helix, Gly residues are fully buried, while the side chains of the Xaa and Yaa residues are largely exposed to solvent, and there is no interaction between three Xaa residues (or three Yaa residues).36 Arg residues were found to have a stabilization effect on collagen triple helices because the guanidinium side chain could form hydrogen bonding interactions to the backbone carbonyl group in a neighboring strand.36,37 Thus, in R2F1 and R2Y1 heterotrimers, the Arg residues not involved in cation−π interactions could interact with the backbone carbonyl groups in the adjacent strand to stabilize the triple helical conformation. In contrast, in R1F2 and R1Y2 heterotrimers, the surplus aromatic residues not participating in cation−π interactions might induce unfavorable interactions with water due to their nonpolar aromatic side chains, leading to less stable constructs. These could explain the observation that the cation−π induced AAB-type heterotrimers prefer R2F1 and R2Y1 configurations. We also conducted simple molecular modeling using Discovery Studio software to determine the peptide chain arrangement within the triple helix of R2F1, R1F2, R2Y1, and R1Y2. The solution structure determined by Hartgerink et al. (PDB entry 2KLW)33 was used as the initial conformation for our modeling. By implanting our sequences into the peptides and performing energy minimization, we obtained the final modeled structures and inspected the cation−π interacting pairs. For each AAB-type heterotrimer, three possible arrangements were tested. For example, the three possible arrangements R−R−F, R−F−R, F−R−R were examined for R2F1. From the modeled structures (Figures S12−S15 of the Supporting Information), we found that potential cation−π interacting pairs exist in an R−F−R or R−Y−R register in R2F1 or R2Y1 while an R−F−F or R−Y−Y register was found in R1F2 or R1Y2. No such cation−π interacting pairs could be found in the other arrangements, suggesting that R−F−R (R2F1), R−Y− R (R2Y1), R−F−F (R1F2), and R−Y−Y (R1Y2) would be the preferred arrangements in these heterotrimers, because they could generate favorable interchain cation−π interactions to stabilize the structure.

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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.5b11189. Peptide sequences and molecular weights; additional kinetic data; HPLC chromatograms; MALDI-TOF mass spectra; additional CD, DSC, and NMR spectra; and molecular modeled structures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +886-3-5715131 ext 35635. Fax: +886-3-5711082. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Taiwan Ministry of Science and Technology (MOST 103-2113-M-007-008) and National Tsing Hua University (104N2011E1). We are grateful to the National Center for High-Performance Computing (NCHC) for computer time and facilities. We also thank Mr. Tang-Chun Kao for his contributions in the early stage of this work.

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REFERENCES

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CONCLUSIONS In the present work, we have demonstrated that the formation of AAB-type heterotrimeric helices can be induced via interchain cation−π interactions and the composition of heterotrimers may be systematically controlled by the relative ratios of mixed cationic and aromatic CMPs in solution. The detailed thermodynamic analysis on synthetic AAB-type collagen heterotrimers by DSC directly showed the contribution of cation−π interactions to the folding of an AAB-type heterotrimer. Kinetic measurements further indicated that the introduced cation−π interactions could accelerate the folding of a heterotrimer. Such analyses reveal new valuable insights into the folding of collagen heterotrimeric helices. This study also extends the applications of cation−π in biomolecular folding and assembly. In conclusion, our results provide a new strategy to assemble small CMPs into AAB-type collagen heterotrimers, which may be used to prepare various collagen heterotrimers for specific studies and applications. F

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DOI: 10.1021/acs.jpcb.5b11189 J. Phys. Chem. B XXXX, XXX, XXX−XXX