Formation of AAB-Type Collagen Heterotrimers ... - ACS Publications

Feb 8, 2017 - were processed using the affiliated Origin software. Samples were prepared in a similar manner to the CD experiments except for that a...
0 downloads 0 Views 1MB Size
Subscriber access provided by University of Newcastle, Australia

Article

Formation of AAB-type collagen heterotrimers from designed cationic and aromatic collagen-mimetic peptides: Evaluation of the C-terminal cation-# interactions Chu-Harn Chiang, Yi-Hsuan Fu, and Jia-Cherng Horng Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01838 • Publication Date (Web): 08 Feb 2017 Downloaded from http://pubs.acs.org on February 11, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 1

Formation of AAB-type collagen heterotrimers from designed cationic and aromatic collagen-mimetic peptides: Evaluation of the C-terminal cation−π interactions

Chu-Harn Chianga, Yi-Hsuan Fua, and Jia-Cherng Horng*ab a

Department of Chemistry, National Tsing Hua University, 101 Sec. 2 Kuang-Fu Rd., Hsinchu, Taiwan 30013, ROC b

Frontier Research Center on Fundamental and Applied Science of

Matters, National Tsing Hua University, 101 Sec. 2 Kuang-Fu Rd., Hsinchu, Taiwan 30013, ROC

*To whom correspondence should be addressed. Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 30013, ROC. phone: +886-3-5715131 ext 35635, fax: +886-3-5711082, e-mail: [email protected]

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 39 Page 2

Abstract Most of natural collagens are heterotrimers composed of two (AAB) or three (ABC) different peptide chains, and thus heterotrimeric constructs are preferable to mimic natural collagens. Exploring the forces to assemble synthetic collagen-mimetic peptides (CMPs) into heterotrimers has been an attractive topic in preparing collagen-related biomaterials. Here we designed and synthesized two cationic CMPs (CR and CK) in which multiple Arg or Lys residues are installed in their C-terminal region, and one aromatic CMP (CF) whose C-terminal end contains multiple Phe residues. Circular dichroism and NMR spectroscopy showed that AAB-type heterotrimers could form in both CR-CF and CK-CF mixtures, suggesting that the C-terminal cation−π interactions between cationic and aromatic residues could serve as a nucleation force and substantially promote the folding of heterotrimers. In particular, only one major heterotrimeric fold was found in each mixture. For CR-CF mixtures, either the heterotrimer with two CR chains and one CF chain or that with one CR chain and two CF chains could form, depending on the molar ratios of CR to CF in solution. By contrast, in CK-CF mixtures only the heterotrimer consisting of two CK chains and one CF chain was found in solution even increasing the ratio of CF, implying that the heterotrimer composed of one CK chain and two CF chains is highly unstable. Additionally, differential scanning calorimetry analysis showed that

ACS Paragon Plus Environment

Page 3 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 3

the folding of these heterotrimers is governed by entropic effects. Together, our results provide a new design to prepare AAB-type collagen heterotrimers and reveal new insights into their folding thermodynamics.

Key words: heterotrimer, cation−π interaction, entropic effect, folding, differential scanning calorimetry

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 39 Page 4

Introduction In humans, collagen accounts for one third of the total protein, and is the most prevalent component of the extracellular matrix. Natural collagens are characterized by extended repeats of X-Y-Gly triplets, where (2S)-proline (Pro) is often found at position X and position Y is frequently occupied by (2S,4R)-4-hydroxyproline (Hyp, O) respectively, making Pro-Hyp-Gly is the most common triplet in collagen.1 Currently, at least 28 types of collagen have been identified and described, and the most abundant type in the human body is type I collagen, which can be commonly found in scar tissue, the end products when tissue heals by repair, as well as tendons, ligaments and bones.2 Collagen is in the form of elongated fibrils, which are composed of triple helices. The three polypeptide chains in a triple helix may be identical (homotrimers) or different (heterotrimers) depending on the collagen type. For type I collagen, it is an AAB-type heterotrimer which makes up from two α1 chains and one α2 chain. The site-specific mutations and alternations of type I collagen can lead to osteogenesis imperfecta and Ehlers-Danlos syndromes, which may affect tissue integrity.2-5 Collagen-mimetic peptides (CMPs) have been widely used as the models to study native collagens due to that animal-derived collagens have may limitations, such as low thermal stability, possible contamination with pathogenic substances, and

ACS Paragon Plus Environment

Page 5 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 5

relative difficulty in the introduction of specific modifications.6-8 In the past decades, homotrimeric models were dominantly used to study natural collagens,2, 7, 8 which might not precisely reflect the properties of heterotrimeric collagens, and thus it would be preferable to use heterotrimeric constructs to investigate this class of collagens. Since type I collagen is the main component of extracellular matrix in vertebrates and defects of type I collagen are associated with tissue malfunctions and diseases, preparation of AAB-type heterotrimers would be an important subject for better understanding type I collagen. Electrostatic interactions were shown to have a great contribution to collagen triple-helix stability,9 implying their great potentials to assist the folding of heterotrimers. Indeed, ion pair interactions have been successfully demonstrated to induce the formation of collagen heterotrimers,10, 11 and computational design of CMPs to form heterotrimers have been also developed based on the electrostatic interactions between chains.12-15 In addition to ion pair interactions, another type of electrostatic interactions, cation−π interactions, are an important but generally underappreciated noncovalent binding force in biological system. Numerous studies have reported the occurrence of cation–π interactions in protein structures16-22 and in protein−ligand interfaces.19, 23, 24 Previously we found that sidechain cation−π interactions between cationic and aromatic residues could confer significant stability to the collagen triple helix,25 and promote the self-assembly of CMPs into

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 39 Page 6

higher-order structure as well.26 Our recent work further showed that cation–π interactions could induce the folding of AAB-type collagen heterotrimers by installing the cationic (Arg) and aromatic (Phe, Tyr) residues into the designed CMPs.27 In that work we could exclusively obtain the heterotrimer composed of two cationic chains and one aromatic chain but failed to prepare the pure heterotrimer with one cationic chain and two aromatic chains. Nonetheless, it opened the possibility to utilize such an interaction to generate various folds of collagen heterotrimers by designing the CMPs with well-defined sequences. Early studies suggested that the C-terminus plays a more critical role than the N-terminus on the trimerization process of a collagen triple helix.28-31 This trimerization processes involve selection, binding and registration of the triple helices and leads the chains to a most stable structure at the end of folding. Brodsky, Baum, and coworkers also showed that the formation of a collagen triple helix initiates at the C-terminus and propagate to the N-terminus.32 However, their later studies indicated that CMPs could fold into a triple helix starting from either the N-terminus or the C-terminus, which depends on where the POG-rich region is located.33, 34 In addition, the work by Bächinger et al. demonstrated that the formation of a collagen triple helix could be initiated at either end depending on the location of a nucleation domain (disulfide knot or foldon).35 These results suggested that installing a nucleation

ACS Paragon Plus Environment

Page 7 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 7

domain at the end of CMPs may assist the formation of heterotrimers. Accordingly, to develop a robust strategy for making collagen heterotrimers, we attempted to improve our design of CMPs and evaluate if the C-terminal cation–π interactions can serve a nucleation force and facilitate the formation of AAB-type collagen heterotrimers. As shown in Figure 1, herein we designed and prepared the CMPs with three cationic (Arg, Lys) or aromatic (Phe) residues in the three C-terminal X-Y-Gly triplets, and intended to generate cation−π interactions from the sidechains and promote the folding of AAB-type heterotrimers. Circular dichroism (CD), differential scanning calorimerty (DSC), and nuclear magnetic resonance (NMR) measurements showed that by mixing the cationic CMPs with the aromatic CMPs we could exclusively obtain one major heterotrimeric species in solution. DSC measurements further indicated that entropic effects dominate the folding of these AAB-type collagen heterotrimers, revealing new insights into the formation of a heterotrimeric helix via cation−π interactions.

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 39 Page 8

Figure 1. (A) Sequences of the designed CMPs: CR, CK, CF. (B) Illustration of the potential C-terminal cation−π interacting pairs in two AAB-type heterotrimeric arrangements, where X is Arg or Lys, and O is Hyp. (C) A simple structure model to show the relative position of Arg (in green) and Phe (in magenta) in the C-terminal region of two different chains. The model was generated with PyMOL v0.99 and the PDB entry 1CAG.36

Materials and Methods Peptide Preparation Chemical reagents and fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids were purchased from Sigma-Aldrich, J. T. Baker, Advanced Chemtech, CreoSalus and used without further purification. The 15N-labeled glycine was obtained from Sigma-Aldrich. All the peptides were synthesized on a 0.06-mmol scale using solid-phase synthesis and Fmoc chemistry on a PS3 peptide synthesizer (Protein Technologies Inc.). Rink amide resin and HBTU-mediated coupling were used for the synthesis. Four equivalents of Fmoc-protected amino acids or Fmoc-Pro-Hyp-Gly-OH were used in the coupling reactions while three equivalents of Fmoc-protected

ACS Paragon Plus Environment

Page 9 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 9

15

N-labeled Gly was used for the coupling. The N-terminus of each peptide was

acetylated before cleavage from the resin. Use of Rink amide resin also generated an amidated C-terminus upon cleavage. After complete synthesis, a solution of either 95% trifluoroacetic acid (TFA)/2.5% triisopropylsilane (TIS)/2.5% H2O (v/v) or 90% TFA/10% anisole (v/v) was used to cleave peptides from the resin and remove the sidechain protecting groups. Crude products were collected by precipitating with cold methyl t-butyl ether. Peptides were purified by reverse-phase high performance liquid chromatography (HPLC) with a YMC semi-preparative C18 column using the gradients of acetonitrile and H2O containing 0.1% (w/v) TFA as counter ions. Purities of the peptides were greater than 90% based on HPLC analysis. Identities of the peptides were confirmed by a matrix-assisted desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Autoflex III Smartbeam LRF-200-CID, Bruker Daltonics). The calculated and observed masses were CR: 2592.29 (calcd.), 2593.28 (obsd. [MH+]); CK: 2508.23 (calcd.), 2509.08 (obsd. [MH+]); CF: 2614.77 (calcd.), 2637.21 (obsd. [MNa+]).

Circular Dichroism (CD) Spectroscopy CD measurements were performed on an Aviv model 410 CD spectrometer, using a 1-mm pathlength quartz cuvette. Samples were dissolved in pH 7.0 and 20

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 39 Page 10

mM sodium phosphate buffer. For the experiments conducted at pH 3.0, the peptides were dissolved in 50 mM acetic acid. Prior to measurements, the peptide solutions were heated at 80 °C for 20 min and followed by cooling down to room temperature for 15 min before incubation at 4 °C for at least 1 day. A peptide concentration of 0.2 mM was used for both homotrimer and heterotrimer measurements. Thermal unfolding curves were obtained by recording ellipticity at 224 or 225 nm in the temperature range of 14 to 60 °C with an average heating rate of 0.16 °C/min and an interval of 1 °C. The values of Tm were determined by taking the first derivative of a melting curve versus temperature and were also estimated by fitting the curves into a simple two-state model.37

Refolding Kinetics In the study of refolding kinetics, the peptide samples were heated at 80 °C for 20 min and then were immediately transferred into a pre-cooled 1-mm cuvette at 4 °C. The CD signals at 224 or 225 nm were monitored for 6 h and the time prior to recording the first data (the dead time) was approximately 50 s. To evaluate refolding rate, refolding half-time (t1/2), the time that half of the peptides require to fold into triple helices, i.e. when the fraction folded (F) equals 0.5, was obtained as an index for comparison of folding rates. The fraction folded was calculated from equation (1),

ACS Paragon Plus Environment

Page 11 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 11

 – 

F =   –  

(1)



where θt is the ellipicity at time t, θf is the ellipicity of the folded form at 4 °C, and θu is the ellipicity of the unfolded form at 4 °C. θf was obtained by measuring the ellipticity of the same sample after at least 24-h incubation at 4 °C, while θu was determined by linear extrapolating the refolding curves to time zero.

Differential Scanning Calorimetry (DSC) DSC measurements were conducted on a MicroCal VP-DSC instrument at National Chung Hsing University Instrument Center and the transition curves were processed using the affiliated Origin software. Samples were prepared in a similar manner to the CD experiments except for that a peptide concentration of 0.4−0.6 mM was used. All DSC samples were degassed at 12 °C for at least 5 min before measurements, and then the profiles were obtained at a heating rate of 0.1 °C /min, from 12 °C to 80 °C. Each curve was subjected to baseline subtraction before data analysis. The Tm values were obtained at the maximum point of transition and values of ∆H were calculated by directly integrating the DSC endotherm. The ∆S values at

Tm was obtained using equation (2), ∆

= ∆.  

(2)

where c is the concentration of monomeric peptides and R is the universal gas

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 39 Page 12

constant. In the analysis, we assumed that ∆Cp is 0 for the triple helix folding and both of ∆H and ∆S are independent of temperature. Values of ∆G were calculated as ∆G = ∆H − T∆S.

Nuclear Magnetic Resonance (NMR) NMR experiments were performed on a Varian VNMRS 700 MHz or a Bruker DMX 600 MHz spectrometer at the National Tsing Hua University Instrumentation Center. 1H,15N-HSQC (heternuclear single quantum coherence) spectra were recorded with a peptide concentration of approximately 0.4 to 0.6 mM, while TOCSY (total correlation spectroscopy) and NOESY (nuclear Overhauser effect spectroscopy) spectra were taken using 1 to 2 mM of peptides. For the measurements at pH 7.0, the samples were dissolved in 90% H2O/10% D2O with 20 mM phosphate buffer. For the experiments at pH 3.0, the samples were dissolved in a solution of 90% H2O/10% D2O containing 50 mM acetic acid. The samples were prepared in a same manner to CD experiments, and were incubated overnight at 4 °C before NMR measurements. 1

H-NMR spectra were internally referenced to 4,4-dimethyl-4-silapentane-1-sulfonic

acid (DSS) sodium salt at 0.0 ppm. A mixing time of 75 ms was used in the TOCSY experiments and 250 ms was used in the NOESY experiments. The 1H,15N-HSQC spectra were recorded at low (10 °C) and high (40 or 50 °C) temperatures to identify

ACS Paragon Plus Environment

Page 13 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 13

the resonances from trimers and the resonances from the monomers. The spectra were analyzed with SPARKY.38

Results and discussion Design of collagen-mimetic peptides to favor the folding of heterotrimers via the C-terminal cation−π interactions Although in the X-Y-Gly repeats of collagen cationic and aromatic residues are not dominant, previous studies showed that cationic residues, such as Lys and Arg, are frequently found in the Y position while aromatic residues have a higher propensity in the X position.39 Our study further found that the cation–π interactions between the sidechains of cationic and aromatic residues could be generated to stabilize the collagen triple helix if cationic residues were placed in the Y position and aromatic residues were installed in the X position.25 These results suggested that it might require to install cationic residues in position Y and aromatic residues in position X to generate an appropriate register and induce interchain cation−π interactions for forming collagen heterotrimers. In fact, our most recent work demonstrated that cation−π interactions did induce the folding of AAB-type heterotrimers.27 In that work, based on the (Pro-Hyp-Gly)9 sequence a cationic (Arg) or an aromatic residue (Phe, Tyr) was incorporated into alternate X-Y-Gly triplets, which we could obtain a

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 39 Page 14

dominant and single form of AAB-type heterotrimer composed of two cationic chains and one aromatic chain : [(POGPYG)4(POG)]2[(POGXOG)4(POG)]1, where X is Phe or Tyr and Y is Arg. However, we failed to produce a pure AAB-type fold with one cationic chain and two aromatic chains. Since the nucleation site is critical and may determine the folding direction of a collagen triple helix,33-35 the installation of cation−π interactions at the end could lead to the formation of heterotrimers. Moreover, according to the importance of the C-terminus on collagen trimerization28-32 and our previous study that showed the C-terminus has a more important contribution on the folding of (POG)8-based CMPs,40 we inferred that installing cation−π interactions into consecutive X-Y-Gly triplets of the C-terminal end might lead to different impacts on the formation of AAB-type collagen heterotrimers. Thus in this work we designed and synthesized three CMPs (CR, CK, and CF) whose sequences are shown in Figure 1 to explore the effects of C-terminal cation−π interactions on the formation of AAB-type heterotrimers. In addition, all of the peptides synthesized in this study have an acetylated N-terminus and an amidated C-terminus to avoid any possible charge repulsions between free amino groups or carboxyl groups. As shown in Figure 1, based on our design we anticipated that the cation−π interactions induced at the C-terminus could serve as a nucleation force and promote

ACS Paragon Plus Environment

Page 15 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 15

the folding of AAB-type heterotrimers when cationic peptides mix with aromatic peptides. Nevertheless, we could not exclude the possibility that the trimerization initiates at the POG-rich N-terminus. In case of folding from the N-terminus, several potential folds may occur when cationic peptides mix with aromatic peptides. For example, the mixture of CR and CF may lead to four possible trimers: (CR)3 homotrimer, (CF)3 homotrimer, (CR)2(CF)1 heterotrimer, and (CR)1(CF)2 heterotrimer if the folding starts from the N-terminus. Thereby, these designed CMPs may also allow us to test if the folding of AAB-type heterotrimers follows a C→N mechanism.

CD measurements reveal the formation of heterotrimers After preparation of the peptides, CD spectroscopy was used to characterize the designed CMPs and measure their conformational stability. All of the peptides exhibit typical polyproline type II (PPII) conformation at 4 °C as shown by the characteristic CD spectrum with a positive peak around 225 nm and an intense negative peak below 210 nm. We monitored the temperature dependent CD signals at 224 or 225 nm to measure the thermal stability for the triple helices formed by the CMPs. As shown in Figure 2, CR and CK can form homotrimers since they display a cooperative thermal transition curve, a characteristic of collagen triple helices. It was reported that the guanidinium group of Arg could form hydrogen bonds with the backbone carboxyl

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 39 Page 16

groups within a triple helix and impose stabilization effects.41, 42 As expected, (CR)3 forms a more stable triple helix than (CK)3. From a two-state model fitting, the Tm value was determined to be 43.2 °C for (CR)3 and 28.8 °C for (CK)3 respectively. By contrast, CF does not form a homotrimer due to the lack of a cooperative unfolding curve. To test if cationic CMPs could wind up with aromatic CMPs to form heterotrimers, we mixed cationic peptides (CR or CK) with aromatic peptides (CF) in a ratio of 2:1 or 1:2, and conducted CD-monitored thermal unfolding measurements. As shown in Figures 2A and 2B, all the mixtures display a cooperative unfolding curve, indicating the formation of triple helices. We used first derivatives of the transition curves versus temperature to determine the melting temperatures and assess the number of trimeric folding types in the CMP mixtures. As shown in Figures 2C and 2D, all of the peptide solutions exhibited one transition, suggesting that only one dominant trimeric species exists in each solution. By taking the first derivatives of the unfolding curves, we determined the Tm value for each mixture, which is 41 °C for 2CR:1CF, 37 °C for 1CR:2CF, 28 °C for 2CK:1CF, and 27 °C for 1CK:2CF respectively. These Tm values are different from that of (CR)3 or (CK)3, which is 44 °C for (CR)3 and 29 °C for (CK)3 determined by taking the first derivatives of their unfolding curves versus temperature, demonstrating the formation of CR-CF and

ACS Paragon Plus Environment

Page 17 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 17

CK-CF heterotrimers. The melting temperatures were also evaluated by using a simple two-state model, which gave a very similar Tm value for each mixture (Table 1), further suggesting that only one major heterotrimeric species existed in solution. The determined Tm values of the mixtures are only slightly lower than (CR)3 or (CK)3 homotrimer, suggesting that these AAB-type heterotrimers have a comparable stability to that of their corresponding homotrimers. According to our previous finding that the heterotrimer containing two aromatic chains would have a lower Tm than that containing only one aromatic chain,27 we assigned the constituent of the heterotrimer to be (CR)2(CF)1 for the mixture of 2CR:1CF, (CR)1(CF)2 for the mixture of 1CR:2CF, and (CK)2(CF)1 for the mixture of 2CK:1CF. For the mixture of 1CK:2CF, its thermal transition curve was somewhat broad and gave an almost identical Tm value to the mixture of 2CK:1CF. We rationalized this observation by assigning (CK)2(CF)1 as the construct in the mixture of 2CK:1CF since the (CK)1(CF)2 trimer is likely be too unstable to form. From CD measurements, we found that only one major species exists in either the CR-CF or CK-CF mixture, strongly suggesting that the folding of these heterotrimers starts from the C-terminus and cation−π interactions should serve a driving force. The homotrimers and heterotrimers formed from the designed CMPs and their corresponding Tm values are compiled in Table 1.

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 39 Page 18

Figure 2. CD-monitored thermal unfolding curves for (A) CR, CF, and CR-CF mixtures, and (B) CK, CF, and CK-CF mixtures. The first derivatives of the thermal unfolding curves versus temperature for (C) CR and CR-CF mixtures, and (D) CK and CK-CF mixtures. All the measurements were carried out using an average heating rate of 0.16 °C /min in the pH 7.0 buffer containing 20 mM sodium phosphate and 0.2 mM peptides.

ACS Paragon Plus Environment

Page 19 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 19

Table 1. Tm values measured by CD for the peptide mixtures in pH 7.0 phosphate buffer.a

Tm (°C) by two-state fitting

Major species

mixtures

Tm (°C) from first derivatives

CR only

44

43.2

(CR)3 homotrimer

CK only

29

28.8

(CK)3 homotrimer

CF only

No triple helix

No triple helix

CF monomer

2CR:1CF

41

40.4

(CR)2(CF)1 heterotrimer

1CR:2CF

37

37.4

(CR)1(CF)2 heterotrimer

2CK:1CF

28

27.3

(CK)2(CF)1 heterotrimer

1CK:2CF

27

27.0

(CK)2(CF)1 heterotrimer

Peptide

a

The deviations for Tm values were within ±1.0 °C.

Formation of heterotrimers verified by NMR spectroscopy To confirm the formation of AAB-type heterotrimers, we prepared 15N-labeled CMPs whose Gly in one X-Y-Gly triplet of the sequence was substituted with 15

N-labeled Gly: (the labeled position is indicated by an asterisk)

(POG)6(PRG*)(PRG)2, (POG)6(PKG*)(PKG)2, (POG)6(FOG*)(FOG)2, and conducted 1H,15N-HSQC measurements. As shown in Figure 3, the overlapped 1

H,15N-HSQC spectra of CR peptides and CR-CF mixtures reveal that new cross

peaks exist in solution in addition to those belonging to (CR)3 trimers, suggesting that the heterotrimers composed of CR and CF form. For the 2CR:1CF mixture (Figure 3A), the new peaks were assigned as the resonances of (CR)2(CF)1 heterotrimer. The 1

H,15N-HSQC spectrum of 1CR:2CF mixture (Figure 3B) is different from that of

2CR:1CF mixture and displays some distinct cross peaks, providing a strong evidence

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 39 Page 20

that the heterotrimer in 1CR:2CF mixture has a different composition. Thus, we assigned those peaks as the resonances of (CR)1(CF)2. It is noted that the NMR experiments for CR-CF mixtures were conducted at pH 3.0 (in 50 mM acetic acid) due to that the CF peptide-containing mixtures with a high concentration in neutral phosphate buffer were not very soluble. To make sure that the heterotrimers fold at pH 3.0 and have a comparable stability to that at pH 7.0, the CD-monitored thermal unfolding curves of the CR-CF mixtures were also obtained at pH 3.0. As shown in Figure S1 of the Supporting Information, the constructs at pH 3.0 exhibited a similar stability to that at pH 7.0. The results demonstrated that the CR-CF mixtures did form heterotrimers at pH 3.0 and the NMR data could be used to represent the results of CR-CF heterotrimers at pH 7.0. To further verify the formation of CR-CF heterotrimers and the existence of cation−π interactions between chains, the NOESY and TOCSY experiments were conducted for 2CR:1CF mixture under the same condition as HSQC experiments. As shown in Figure S2 of the Supporting Information, the NOE signals between the aromatic hydrogens of Phe and the sidechain hydrogens of Arg were observed, suggesting that Arg and Phe are interacting within the triple helix. The NOE results, therefore, complemented the 1

H,15N-HSQC data and provided a strong evidence for the presence of cation−π

interactions in the collagen heterotrimers of (CR)2(CF)1 and (CR)1(CF)2.

ACS Paragon Plus Environment

Page 21 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 21

Figure 3. Overlapped 1H,15N-HSQC of (A) CR (green) and 2CR:1CF (red) and (B) CR (green) and 1CR:2CF (red). The spectra were recorded at pH 3.0 and 10 °C. The “t” denotes trimer and “m” denotes monomer.

In a similar manner, we also performed NMR experiments for CK-CF mixtures at pH 7.0. As shown in Figure 4, new cross peaks are observed in the overlapped CK and CK-CF mixture 1H,15N-HSQC spectra, indicating the existence of CK-CF heterotrimers. Strikingly, we found that both the 2CK:1CF and 1CK:2CF mixtures

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 39 Page 22

exhibited an almost identical 1H,15N-HSQC spectrum, which was consistent with the CD measurements that gave a same Tm value for these two mixtures, supporting our argument that the same major heterotrimer (CK)2(CF)1 forms in these two mixtures. Therefore, we assigned the new peaks as the resonances of (CK)2(CF)1. According to CD and NMR measurements, we did not observe the heterotrimer of (CK)1(CF)2 although we increased the molar ratio of CF peptides in solution, again suggesting that (CK)1(CF)2 might be too unstable to form. Likewise, the NOESY and TOSCY spectra were also recorded for 2CK:1CF mixture to inspect the cation−π interactions between the sidechains of Lys and Phe. As shown in Figure S3 of the Supporting Information, the NOE signals could be found between the aromatic hydrogens of Phe and the sidechain hydrogens of Lys, supporting our argument that Lys and Phe interacts within a heterotrimer. Additionally, to test the hypothesis that cation−π interactions are the force to drive the assembly of AAB-type heterotrimers, we further measured the 1

H,15N-HSQC spectra at high pH values for the 2CK:1CF mixture. Although we could

not obtain a good signal-to-noise spectrum at the pH greater than 11 due to the concentrated salts at high pH values, we could still observe the loss of cation−π interactions at pH 11.0. As shown in Figure S4 of the Supporting Information, we could observe the resonance of (CK)3 homotrimer in the HSQC spectrum at pH 11.0,

ACS Paragon Plus Environment

Page 23 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 23

which was not found at pH 7.0 (Figure 4A), indicating that weakening the cation−π interactions between Lys and Phe at the pH around the pKa of Lys leads to the formation of (CK)3. Moreover, the signal of (CK)2(CF)1 at pH 11.0 is weaker than that at pH 7.0. The results provide strong evidence to support our thesis that cation−π interactions are the driving force to fold heterotrimers.

Figure 4. Overlapped 1H,15N-HSQC of (A) CK (green) and 2CK:1CF (red) and (B) CK (green) and 1CK:2CF (red). The spectra were recorded at pH 7.0 and 10 °C. The “t” denotes trimer and “m” denotes monomer.

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 39 Page 24

DSC analyses provide the thermodynamic properties of heterotrimers To better understand of the thermodynamic features of CR-CF and CK-CF heterotrimers, we conducted DSC measurements to obtain their detailed folding thermodynamic parameters. As shown in Figure 5, the DSC thermograms of the heterotrimeric helices (CR)2(CF)1, (CR)1(CF)2, and (CK)2(CF)1 all exhibit only one single transition, concurring with the observations by CD and NMR and again indicating that these heterotrimers are the dominant species in solution. DSC analyses also support that the folding direction of heterotrimers is from the C-terminus to the N-terminus. By integrating the area under the DSC curves, we could obtain the enthalpy change upon unfolding a triple helix and calculate the thermodynamic parameters of the transition from triple helices to monomeric states, as described in the literature.43-46 From DSC analysis, we found that the homotrimers (CR)3 and (CK)3 have a much more favorable folding enthalpic value (∆H) than each of their corresponding heterotrimers although the homotrimers and heterotrimers have a similar melting temperature as shown in Table 2. We also chose an intermediate temperature among the Tm values of the triple helices as a reference temperature, which would give the most accurate thermodynamic parameters,45 to calculate folding entropy (T∆S) for the homotrimers and heterotrimers. In this case, 41 °C was used for (CR)3 and its corresponding heterotrimers while 28 °C was used for (CK)3 and its

ACS Paragon Plus Environment

Page 25 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 25

corresponding heterotrimer. As shown in Table 2, compared to (CR)3 or (CK)3, the heterotrimers have a much more favorable T∆S value upon folding, indicating that the folding of (CR)2(CF)1, (CR)1(CF)2, and (CK)2(CF)1 is driven by entropic effects. Interestingly, while comparing the folding entropy of (CR)3 homotrimer with its corresponding heterotrimers [(CR)2(CF)1 and (CR)1(CF)2], we can find that the T∆S value becomes more positive when the fraction of CF increases in the triple helix. This strongly shows that the water molecules ordered around the hydrophobic Phe residues on CF peptides will release upon folding into triple helices with CR peptides, leading to the significant increase in entropy. As shown in Table 2, the results are different from what we observed for the previously designed R3 homotrimer and R2F1 heterotrimer whose R is (POGPRG)4(POG) and F is (POGFOG)4(POG), where the folding of R2F1 is driven by enthalpic effects.27 In R2F1, the Arg and Phe residues are incorporated into the peptide in alternate Pro-Hyp-Gly triplets while these two residues are inserted into three consecutive C-terminal Pro-Hyp-Gly triplets of the peptides for (CR)2(CF)1 and (CR)1(CF)2 heterotrimers. This finding suggests that the highly dense Phe residues at the C-terminus of the CF peptide may cause more considerable water ordering than the relatively dispersed Phe residues within the F peptide, and thus induces more entropy release when the aromatic peptides wind up with the cationic peptides to form trimers. For (CK)3 and (CK)2(CF)1, the folding of

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 39 Page 26

(CK)2(CF)1 also induces a positive entropy change while the formation of (CK)3 causes entropy decrease, showing that the formation of (CK)2(CF)1 is also entropy driven. Our results from the previous design of R2F1heterotrimer and current design of (CR)2(CF)1, (CR)1(CF)2, and (CK)2(CF)1 heterotrimers reveal an important piece of information that their folding thermodynamics is different although cation−π interactions are suggested to play the critical role in the formation of these heterotrimers. This implies that the thermodynamic features for cation−π induced AAB-type heterotrimers may depend on the interacting position and the dispersion of interacting pairs within a triple helix. CD results and DSC analyses both showed that mixing one stable CMP (CR or CK) with one unstable CMP (CF) could help the formation of heterotrimers whose stability is slightly lower or similar to their corresponding cationic homotrimer ((CR)3 or (CK)3). Such a phenomenon was also observed by an early study using a heterotrimeric type IV collagen model peptide to form AAB-type trimers, in which additional hydrogen bonds were formed after mixing the stable and unstable CMPs to stabilize the heterotrimer.47 In our current design of AAB-type heterotrimers, the situation is different because no extra hydrogen bonds would form while mixing cationic and aromatic peptides to fold heterotrimers. Thus, we believe that cation−π interactions are the key factor to drive assembly of

ACS Paragon Plus Environment

Page 27 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 27

heterotrimers. While comparing the CR-CF mixture with the CK-CF mixture, we learned that either (CR)2(CF)1 or (CR)1(CF)2 heterotrimer can be produced depending on the mixing ratios of CR to CF but only (CK)2(CF)1 heterotrimer can form in CK-CF solution. This might be rationalized by considering the strength of cation−π interactions in these two cation-aromatic pairs. Since the guanidinium sidechain of Arg is less hydrated than the amino sidechain of Lys in aqueous solution, Arg incurs less desolvation penalty and forms a stronger cation−π interaction with Phe than does Lys. This was also found in our previous study on the contribution of cation−π interactions to the collagen triple helix stability, in which the Arg-Phe pair has a larger stabilization effect than the Lys-Phe pair.25 Therefore, the strength of cation−π interaction between Lys and Phe could be not enough to support the formation of (CK)1(CF)2. In addition, compared to the design of introducing cation-aromatic pairs alternately within the CMPs, the insertion of consecutive cationic or aromatic residues into the C-terminal end of a CMP seems more successful at preparing a pure AAB-type heterotrimeric species because the R-F mixture failed to produce a single component of R1F2 heterotrimer in the mixture. Since the CD and DSC data showed that the folding of heterotrimers initiates at the C-terminus, it is likely that the multiple C-terminal cation−π pairs generate a strong force to significantly affect the

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 39 Page 28

arrangements of CMPs and determine the folding type when the triple helix starts to form from the C-terminal end. This may explain why the CR-CF and CK-CF mixtures could produce a single AAB-type heterotrimeric component at different mixing ratios while the R-F mixture could not.

Figure 5. DSC profiles for (A) (CR)3, (CR)2(CF)1, (CR)1(CF)2 and (B) (CK)3, (CK)2(CF)1. All the measurements were conducted in pH 7.0 and 20 mM phosphate buffer with a peptide concentration of 0.4 − 0.6 mM.

ACS Paragon Plus Environment

Page 29 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 29

Table 2. Thermodynamic data of the trimer folding derived from DSC analysis, and refolding half-time of the triple helices monitored by CD. Triple helix (CR)3 (CR)2(CF)1 (CR)1(CF)2

Tm (°C) 42.6 40.9 40.2

∆H (kJ/mol) −T∆S (kJ/mol)a ∆G (kJ/mol)

t1/2 (min)

-73.2

33.3

-39.9

32

-61.3

21.8

-39.5

38

-20.5

-21.2

-41.2

23

(CK)3

27.5

-53.7

15.9

-37.3

34

(CK)2(CF)1

28.1

-26.4

-11.4

-38.2

35

R3b

48.7

-49.5

8.94

-40.5

>500

R2F1b a

45.3

-61.8

21.8

-39.9

193

The temperature used for the calculation of T∆S is 41 °C for (CR)3, (CR)2(CF)1,

(CR)1(CF)2, and 28 °C for (CK)3, (CK)1(CF)2. The data of R3 and R2F1 are from reference 27.

b

Kinetic measurements and ionic strength dependent evaluation on heterotrimers We also performed refolding kinetic measurements for all the heterotrimers and homotrimers and used t1/2, which is the time required to refold 50% triple helices, to compare their folding rates. The measured refolding curves are shown in Figure S5 of the Supporting Information. As shown in Table 2, (CR)2(CF)1 and (CR)1(CF)2 have a similar t1/2 to that of (CR)3, while the t1/2 values of (CK)3 and (CK)2(CF)1 are almost identical, indicating that the heterotrimers refold with a comparable rate to their corresponding homotrimers. Our previous design of AAB-type collagen heterotrimer R2F1, in which the cation−π interaction occurs every two triplets, showed that R2F1 refolded much faster than its corresponding cationic homotrimer R3 (Table 2) and

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 39 Page 30

refolding kinetics played an important role controlling the formation of R2F1.27 By contrast, the heterotrimers designed in this work all refolds in a similar rate to that of their corresponding cationic homotrimers, suggesting that the C-terminal cation−π interactions do not significantly speed up the refolding of heterotrimers and kinetic factors are not crucial to facilitate the formation of (CR)2(CF)1, (CR)1(CF)2, (CK)2(CF)1 heterotrimers. Furthermore, we investigated the effects of ionic strength on the stability of heterotrimers by adding 150 mM NaCl into the solution. As shown in Figure S6 of the supporting information, we found that the Tm values of (CR)2(CF)1, (CR)1(CF)2, and (CK)2(CF)1 in the presence of 150 mM NaCl are almost identical to those measured without NaCl. The results are similar to what we found for the heterotrimer of R2F1,27 and again show that cation−π interaction induced collagen heterotrimers are not sensitive to ionic strength, making cation−π interaction a robust force for the preparation of AAB-type collagen heterotrimers.

Conclusions In the present study, we have designed two cationic CMPs (CR and CK) and one aromatic CMP (CF) and used CD, NMR, and DSC to demonstrate that AAB-type

ACS Paragon Plus Environment

Page 31 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 31

collagen heterotrimers can be induced to form via cation−π interactions on the C-end of a triple helix. The experimental results also provide the evidence that their folding should follow a C→N mechanism. The heterotrimeric folds between CR and CF could be controlled by the mixing molar ratios of cationic and aromatic CMPs, in particular we could exclusively obtain one dominant heterotrimer for each case in solution. In the case of CK and CF mixtures, although we increased the amount of CF, only one major heterotrimeric fold, (CK)2(CF)1, could be observed due to the low stability of (CK)1(CF)2. The thermodynamic analysis by DSC revealed that entropic effects are the main factor to favor the folding of these heterotrimers, providing new thermodynamic insights into another class of AAB-type collagen heterotrimers that are induced via cation−π interactions. In conclusion, the C-terminal cation−π interactions have been shown an effective force to assist the formation of a pure fold of AAB-type collagen heterotrimer, exhibiting the application potentials of cation−π interactions in preparing collagen heterotrimers for collagen related studies.

Supporting Information TOCSY and NOESY spectra, additional 1H,15N-HSQC spectra, CD-monitored refolding curves, and additional CD-monitored thermal transition curves are included. This material is available free charges via the Internet at http://pubs.acs.org.

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 39 Page 32

Acknowledgments This work was supported by Taiwan Ministry of Science and Technology (MOST 105-2113-M-007-018) and National Tsing Hua University (105N501CE1).

Notes The authors declare no competing financial interest.

References (1) Ramshaw, J. A. M.; Shah, N. K.; Brodsky, B. Gly-X-Y tripeptide frequencies in collagen: A context for host–guest triple-helical peptides. J. Struct. Biol. 1998, 122, 86-91. (2) Shoulders, M. D.; Raines, R. T. Collagen structure and stability. Annu. Rev.

Biochem. 2009, 78, 929-958. (3) Besio, R.; Forlino, A. Treatment options for osteogenesis imperfecta. Expert

Opin. Orphan Drugs 2015, 3, 165-181. (4) Nuytinck, L.; Freund, M.; Lagae, L.; Pierard, G. E.; Hermanns-Le, T.; De Paepe, A. Classical Ehlers-Danlos syndrome caused by a mutation in type I collagen. Am. J.

Hum. Genet. 2000, 66, 1398-1402. (5) Ward, L. M.; Lalic, L.; Roughley, P. J.; Glorieux, F. H. Thirty-three novel

ACS Paragon Plus Environment

Page 33 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 33

COL1A1 and COL1A2 mutations in patients with osteogenesis imperfecta types I-IV.

Hum. Mutat. 2001, 17, 434-434. (6) Nemoto, T.; Horiuchi, M.; Ishiguro, N.; Shinagawa, M. Detection methods of possible prion contaminants in collagen and gelatin. Arch. Virol 1999, 144, 177-184. (7) Engel, J.; Bachinger, H. P. Structure, stability and folding of the collagen triple helix. Top. Curr. Chem. 2005, 247, 7-33. (8) Brodsky, B.; Persikov, A. V. Molecular structure of the collagen triple helix. Adv.

Protein Chem. 2005, 70, 301-339. (9) Persikov, A. V.; Ramshaw, J. A. M.; Kirkpatrick, A.; Brodsky, B. Electrostatic interactions involving lysine make major contributions to collagen triple-helix stability. Biochemistry 2005, 44, 1414-1422. (10) Fallas, J. A.; O'Leary, L. E. R.; Hartgerink, J. D. Synthetic collagen mimics: self-assembly of homotrimers, heterotrimers and higher order structures. Chem. Soc.

Rev. 2010, 39, 3510-3527. (11) Jalan, A. A.; Hartgerink, J. D. Pairwise interactions in collagen and the design of heterotrimeric helices. Curr. Opin. Chem. Biol. 2013, 17, 960-967. (12) Persikov, A. V.; Ramshaw, J. A. M.; Kirkpatrick, A.; Brodsky, B. Peptide investigations of pairwise interactions in the collagen triple-helix. J. Mol. Biol. 2002,

316, 385-394.

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 39 Page 34

(13) Fallas, J. A.; Hartgerink, J. D. Computational design of self-assembling register-specific collagen heterotrimers. Nat. Commun. 2012, 3, 1087. (14) Giddu, S.; Xu, F.; Nanda, V. Sequence recombination improves target specificity in a redesigned collagen peptide ABC-type heterotrimer. Proteins 2013, 81, 386-393. (15) Jalan, A. A.; Demeler, B.; Hartgerink, J. D. Hydroxyproline-Free Single Composition ABC Collagen Heterotrimer. J. Am. Chem. Soc. 2013, 135, 6014-6017. (16) Burley, S. K.; Petsko, G. A. Amino-aromatic interactions in proteins. FEBS Lett. 1986, 203, 139-143. (17) Flocco, M. M.; Mowbray, S. L. Planar stacking interactions of arginine and aromatic side-chains in proteins. J. Mol. Biol. 1994, 235, 709-717. (18) Mitchell, J. B. O.; Nandi, C. L.; McDonald, I. K.; Thornton, J. M.; Price, S. L. Amino/aromatic interactions in proteins: Is the evidence stacked against hydrogen bonding? J. Mol. Biol. 1994, 239, 315-331. (19) Ma, J. C.; Dougherty, D. A. The cation-π interaction. Chem. Rev. 1997, 97, 1303-1324. (20) Gallivan, J. P.; Dougherty, D. A. Cation-π interactions in structural biology. Proc.

Natl. Acad. Sci. USA 1999, 96, 9459-9464. (21) Minoux, H.; Chipot, C. Cation−π interactions in proteins:  Can simple models provide an accurate description? J. Am. Chem. Soc. 1999, 121, 10366-10372.

ACS Paragon Plus Environment

Page 35 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 35

(22) Mahadevi, A. S.; Sastry, G. N. Cation-π interaction: Its role and relevance in chemistry, biology, and material science. Chem. Rev. 2013, 113, 2100-2138. (23) Meyer, E. A.; Castellano, R. K.; Diederich, F. Interactions with aromatic rings in chemical and biological recognition. Angew. Chem. Int. Ed. 2003, 42, 1210-1250. (24) Biot, C.; Buisine, E.; Rooman, M. Free-energy calculations of protein−ligand cation−π and amino−π interactions: From vacuum to proteinlike environments. J. Am.

Chem. Soc. 2003, 125, 13988-13994. (25) Chen, C.-C.; Hsu, W.; Hwang, K.-C.; Hwu, J. R.; Lin, C.-C.; Horng, J.-C. Contributions of cation-π interactions to the collagen triple helix stability. Arch.

Biochem. Biophys. 2011, 508, 46-53. (26) Chen, C.-C.; Hsu, W.; Kao, T.-C.; Horng, J.-C. Self-assembly of short collagen-related peptides into fibrils via cation-π interactions. Biochemistry 2011, 50, 2381-2383. (27) Chiang, C.-H.; Horng, J.-C. Cation−π interaction induced folding of AAB-type collagen heterotrimers. J. Phys. Chem. B 2016, 120, 1205-1211. (28) Fessler, L. I.; Fessler, J. H. Protein assembly of procollagen and effects of hydroxylation. J. Biol. Chem. 1974, 249, 7637-7646. (29) Bächinger, H. P.; Fessler, L. I.; Timpl, R.; Fessler, J. H. Chain assembly intermediate in the biosynthesis of type III procollagen in chick embryo blood vessels.

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 39 Page 36

J. Biol. Chem. 1981, 256, 13193-9. (30) Doege, K. J.; Fessler, J. H. Folding of carboxyl domain and assembly of procollagen I. J. Biol. Chem. 1986, 261, 8924-35. (31) Dion, A. S.; Myers, J. C. COOH-terminal propeptides of the major human procollagens. J. Mol. Biol. 1987, 193, 127-143. (32) Bhate, M.; Wang, X.; Baum, J.; Brodsky, B. Folding and conformational consequences of glycine to alanine replacements at different positions in a collagen model peptide. Biochemistry 2002, 41, 6539-6547. (33) Buevich, A. V.; Silva, T.; Brodsky, B.; Baum, J. Transformation of the mechanism of triple-helix peptide folding in the absence of a C-terminal nucleation domain and its implications for mutations in collagen disorders. J. Biol. Chem. 2004,

279, 46890-46895. (34) Hyde, T. J.; Bryan, M. A.; Brodsky, B.; Baum, J. Sequence dependence of renucleation after a Gly mutation in model collagen peptides. J. Biol. Chem. 2006,

281, 36937-36943. (35) Frank, S.; Boudko, S.; Mizuno, K.; Schulthess, T.; Engel, J.; Bächinger, H. P. Collagen triple helix formation can be nucleated at either end. J. Biol. Chem. 2003,

278, 7747-7750. (36) Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M. Crystal and molecular structure

ACS Paragon Plus Environment

Page 37 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 37

of a collagen-like peptide at 1.9 Å resolution. Science 1994, 266, 75-81. (37) Engel, J.; Chen, H.-T.; Prockop, D. J.; Klump, H. The triple helix⇌coil conversion of collagen-like polytripeptides in aqueous and nonaqueous solvents. Comparison of the thermodynamic parameters and the binding of water to (L-Pro-L-Pro-Gly)n and (L-Pro-L-Hyp-Gly)n. Biopolymers 1977, 16, 601-622. (38) Goddard, T. D.; Kneller, D. G. SPARKY 3, University of California, San Francisco, CA: 2008. (39) Persikov, A. V.; Ramshaw, J. A. M.; Kirkpatrick, A.; Brodsky, B. Amino acid propensities for the collagen triple-helix. Biochemistry 2000, 39, 14960-14967. (40) Chen, Y.-S.; Chen, C.-C.; Horng, J.-C. Thermodynamic and kinetic consequences of substitutig glycine at different positions in a Pro-Hyp-Gly repeat collagen model peptide. Biopolymers (Pept. Sci.) 2011, 96, 60-68. (41) Borders, C. L.; Broadwater, J. A.; Bekeny, P. A.; Salmon, J. E.; Lee, A. S.; Eldridge, A. M.; Pett, V. B. A structural role for arginine in proteins: Multiple hydrogen bonds to backbone carbonyl oxygens. Protein Sci. 1994, 3, 541-548. (42) Radmer, R. J.; Klein, T. E. Severity of osteogenesis imperfecta and structure of a collagen-like peptide modeling a lethal mutation site. Biochemistry 2004, 43, 5314-5323. (43) Nishi, Y.; Doi, M.; Doi susumu uchiyama, M.; Nishiuchi, Y.; Nakazawa, T.;

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 39 Page 38

Ohkubo, T.; Kobayashi, Y. Stabilization mechanism of triple helical structure of collagen molecules. Lett. Pept. Sci. 2003, 10, 533-537. (44) Nishi, Y.; Uchiyama, S.; Doi, M.; Nishiuchi, Y.; Nakazawa, T.; Ohkubo, T.; Kobayashi, Y. Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix. Biochemistry 2005, 44, 6034-6042. (45) Shoulders, M. D.; Kotch, F. W.; Choudhary, A.; Guzei, I. A.; Raines, R. T. The aberrance of the 4S diastereomer of 4-hydroxyproline. J. Am. Chem. Soc. 2010, 132, 10857-10865. (46) Shoulders, M. D.; Satyshur, K. A.; Forest, K. T.; Raines, R. T. Stereoelectronic and steric effects in side chains preorganize a protein main chain. Proc. Natl. Acad.

Sci. U.S.A. 2010, 107, 559-564. (47) Xiao, J.; Sun, X.; Madhan, B.; Brodsky, B.; Baum, J. NMR studies demonstrate a unique AAB composition and chain register for a heterotrimeric type IV collagen model peptide containing a natural interruption site. J. Biol. Chem. 2015, 290, 24201-24209.

ACS Paragon Plus Environment

Page 39 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Page 39

Table of Contents Graphic

ACS Paragon Plus Environment