Effect of the -Gly-3(S)-hydroxyprolyl-4(R)-hydroxyprolyltripeptide unit on the stability of collagen model peptides Kazunori Mizuno1, David H Peyton2, Toshihiko Hayashi3, Jurgen Engel4 and Hans Peter ă Bachinger1,5 ă Research Department, Shriners Hospital for Children, Portland, OR, USA Department of Chemistry, Portland State University, Portland, OR, USA Faculty of Pharmaceutical Science, Teikyo Heisei University, Chiba, Japan Biozentrum, University of Basel, Switzerland Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR, USA Keywords 3-hydroxylation; collagen; peptide; posttranslational modification; thermal stability Correspondence H P Bachinger, Research Department, ă Shriners Hospital for Children, 3101 SW Sam Jackson Park Road, Portland, OR 97239, USA Fax: +1 503 221 3451 Tel: +1 503 221 3433 E-mail: hpb@shcc.org Website: http://www.shcc.org/bach_lab.htm (Received 31 July 2008, revised 18 September 2008, accepted 25 September 2008) doi:10.1111/j.1742-4658.2008.06704.x In order to evaluate the role of 3(S)-hydroxyproline [3(S)-Hyp] in the triple-helical structure, we produced a series of model peptides with nine tripeptide units including 0–9 3(S)-hydroxyproline residues The sequences are H-(Gly-Pro-4(R)Hyp)l-(Gly-3(S)Hyp-4(R)Hyp)m-(Gly-Pro-4(R)Hyp)nOH, where (l, m, n) = (9, 0, 0), (4, 1, 4), (3, 2, 4), (3, 3, 3), (1, 7, 1) and (0, 9, 0) All peptides showed triple-helical CD spectra at room temperature and thermal transition curves Sedimentation equilibrium analysis showed that peptide H-(Gly-3(S)Hyp-4(R)Hyp)9-OH is a trimer Differential scanning calorimetry showed that replacement of Pro residues with 3(S)Hyp residues decreased the transition enthalpy, and the transition temperature increases by 4.5 °C from 52.0 °C for the peptide with no 3(S)Hyp residues to 56.5 °C for the peptide with nine 3(S)Hyp residues The refolding kinetics of peptides H-(Gly-3(S)Hyp-4(R)Hyp)9-OH, H-(Gly-Pro-4(R)Hyp)9OH and H-(Gly-4(R)Hyp-4(R)Hyp)9-OH were compared, and the apparent reaction orders of refolding at 10 °C were n = 1.5, 1.3 and 1.2, respectively Replacement of Pro with 3(S)Hyp or 4(R)Hyp has little effect on the refolding kinetics This result suggests that the refolding kinetics of collagen model peptides are influenced mainly by the residue in the Yaa position of the -Gly-Xaa-Yaa- repeated sequence The experiments indicate that replacement of a Pro residue by a 3(S)Hyp residue in the Xaa position of the -Gly-Xaa-4(R)Hyp- repeat of collagen model peptides increases the stability, mainly due to entropic factors The collagen triple helix is probably the most abundant protein motif in the human body It comprises three left-handed polyproline II-like helices with a Gly-Xaa-Yaa repeat These form a right-handed super helix with a one-residue stagger [1,2] The collagen triple helix has many unique properties One of them is the requirement for many post-translational modifications to produce the final tissue form of the molecule [3] In vertebrate collagens, most of the Pro residues in the Yaa position of the -Gly-Xaa-Yaarepeat sequence are nearly completely 4-hydroxylated to 4(R)-hydroxyproline [4(R)Hyp] by the enzyme prolyl 4-hydroxylase (EC 1.14.11.2) This modification in the Yaa position is strongly related to the stability of the collagen triple helix Prolyl 4-hydroxylation also occurs in the Xaa position in invertebrates In addition to prolyl 4(R)-hydroxylation, a small numbers of proline residues are modified to 3(S)-hydroxyproline [3(S)Hyp] [4,5] in many types of vertebrate collagens, such as types I, II, III, IV, V and X Invertebrate collagens also contain 3(S)Hyp, for example interstitial and cuticle collagens of annelids [6], crab sub-cuticular Abbreviations CRTAP, cartilage-associated protein; DSC, differential scanning calorimetry; Hyp, hydroxyproline; P3H1, prolyl 3-hydroxylase 5830 FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS K Mizuno et al Recently, we analyzed the crystal structure of a triplehelical peptide with two 3(S)Hyp residues per chain, i.e H-(Gly-Pro-4(R)Hyp)3-(Gly-3(S)Hyp-c4(R)Hyp)2-(GlyPro-4(R)Hyp)4-OH [29] The backbone of this peptide is almost identical to that of triple-helical peptides comprising the repeated sequences Gly-Pro-Pro and GlyPro-4(R)Hyp in a left-handed ⁄ helical symmetry [32] This finding led us to re-evaluate the data obtained using the peptides acetyl-(Gly-3(S)Hyp-4(R)Hyp)10NH2 and acetyl-(Gly-Pro-4(R)Hyp)3-Gly-3(S)Hyp4(R)Hyp-(Gly-Pro-4(R)Hyp)4-Gly-Gly-NH2 [33] The peptide acetyl-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 did not show any evidence of forming a triple helix when analyzed by sedimentation-equilibrium, CD or NMR analysis [33] We repeated the synthesis of this peptide and also produced several other peptides with 3(S)Hyp in the Xaa position All of these newly synthesized peptides formed a triple-helical structure We confirmed that a peptide with 3(S)Hyp in the Yaa position, acetyl-(GlyPro-3(S)Hyp)10-NH2, does not fold into a triple-helical structure We analyzed the peptides by CD, NMR and differential scanning calorimetry (DSC) to evaluate the effect of 3(S)-hydroxylation on the stability and refolding kinetics of the collagen triple helix Results The CD spectra of the newly synthesized peptide Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 in water shown in Fig is similar to that of other collagen-like peptides The spectrum shows a positive peak around 225 nm °C 80 °C × collagen [7], lobster sub-cuticular membrane collagen [7], squid skin collagen [7], abalone muscle collagen [7], octopus skin collagen [8], octopus arm collagen [9] and jellyfish mesogloea collagen [10] Most earlier reports on 3(S)Hyp depended on amino acid analysis of peptide fragments from crude extracts or whole tissues Only a few studies have determined the position of 3(S)Hyp by amino acid sequencing, and the 3(S)Hyp was found in a -Gly-3Hyp-4Hyp-Gly- sequence in all instances Prolyl 3-hydroxylation is catalyzed by the enzyme prolyl-3-hydroxylase (P3H1, EC 1.14.11.7), which has three family members in vertebrates, but characterization of these enzymes is very limited [11– 13] The analysis of the 3(S)Hyp content is not straightforward [14] 3(S)Hyp degrades much faster than 4(R)Hyp during hydrolysis in m HCl, as used in amino acid analysis [14] In fact, reported contents of 3(S)Hyp are inconsistent even for the same tissue and species [15–17] This is not just due to the heterogeneity of the modification, but also to differences in sample preparation for amino acid analysis As a result of this, some reports may have underestimated the 3(S)Hyp content Type I collagen, which consists of two a1 chains and one a2 chain, has a single 3(S)Hyp residue per chain [18–20] The proline residue at position 986 in the a1 chain is modified to 3(S)Hyp [21] by the protein complex P3H1 ⁄ CRTAP ⁄ cyclophilin B [13] Post-translational modifications are changed in heritable disorders due to mutation and ⁄ or deletion of the enzymes, or due to over-modification, as in osteogenesis imperfecta and Ehlers–Danlos syndrome type VI [22] The 4(R)-hydroxylation in the Yaa position has been well documented as stabilizing the triple-helical structure [23–25] Raines and colleagues [23] synthesized fluoroprolyl compounds containing C-F bonds, a very weak hydrogen bond acceptor [26], in order to determine the mechanism of stabilization The increase in stability is due to a stereoelectronic effect (reviewed in [23]) The effect of 4(R)Hyp in the Xaa position of the -Gly-Xaa-Yaa- collagen sequence has also been analyzed [23,27,28] Compared to the post-translational modification at the C4 position, the effect of 3-hydroxylation of prolyl residues in the collagen helix on its stability has not yet been thoroughly analyzed [29–31] Whether the 3(S)Hyp residue in the Xaa position stabilizes or destabilizes the collagen helix is still controversial In host-guest peptides, it was found that the stability of the triple helix is decreased when Pro in the Xaa position is replaced by either 3(S)Hyp or 3(S)fluoroproline [30,31] It is not possible for 3(S)Hyp to be located in the Yaa position in the triple-helical structure due to steric clashes [31] 3-hydroxyproline in the collagen triple helix 80 °C °C Fig CD spectra of acetyl-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 CD spectra were measured at 4, 20, 40 and 80 °C in water at a concentration of 100 lM The positive ellipticity at 225 nm decreases as the temperature is increased from to 20, 40 and 80 °C FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS 5831 3-hydroxyproline in the collagen triple helix K Mizuno et al × and a negative peak around 196 nm at °C The ellipticity at 225 nm of peptide Ac-(Gly-3(S)Hyp4(R)Hyp)10-NH2 is between that of peptide Ac-(GlyPro-Pro)10-NH2 and Ac-(Gly-Pro-4(R)Hyp)10-NH2, and larger than the other collagen-like peptides which not form a triple helix, Ac-(Gly-4(R)Hyp-Pro)10NH2 and Ac-(Gly-Pro-3(S)Hyp)10-NH2 [33] The temperature scan monitored at 225 nm shows a cooperative transition curve for peptide Ac-(Gly3(S)Hyp-4(R)Hyp)10-NH2 (Fig 2) The ellipticity of the peptide was positive even after the transition at 95 °C Therefore, the characteristics of peptide Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 are similar to those of peptide Ac-(Gly-4(R)Hyp-4(R)Hyp)10-NH2 [34] In order to verify that this transition curve is due to the transition from triple helix to coil, the oligomerization state of the 3(S)Hyp-containing peptides was analyzed by equilibrium sedimentation (Fig S1) Analysis of peptide Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 showed a molecular mass of 8.75 ± 0.15 · 103 Da; the calculated molecular mass of the trimer peptide is 8676 Da We also analyzed H-(Gly-3(S)Hyp-4(R) Hyp)9-OH The molecular mass for this peptide is 8.26 ± 0.21 · 103 Da, which is 7% larger than the calculated trimeric peptide value of 7703 Da, suggest- Temperature (°C) Fig Thermal transition curves of collagen-like peptides The peptides were measured in water and the CD signal was monitored at 225 nm as a function of increasing temperature The peptide concentration was 100 lM and the temperature scanning rate was 10 °CỈh)1 Results are shown for Ac-(Gly-3(S)Hyp-4(R) Hyp)10-NH2 (square), Ac-(Gly-Pro-Pro)10-NH2 (circle), Ac-(Gly-Pro-4(R)-Hyp)10-NH2 (upwards triangle), Ac-(Gly-4(R)Hyp-Pro)10-NH2 (downwards triangle) and Ac-(Gly-Pro-3(S)Hyp)10-NH2 (diamond) All data except those for Ac-(Gly-3(S)Hyp-4(R) Hyp)10-NH2 and Ac-(Gly-Pro-3(S)Hyp)10-NH2 are from a previous study [34] and are included as a reference 5832 ing that most of the peptide is trimeric and that some aggregates are in solution at 25 °C We conclude from these experiments that the collagen model peptide with repeated tripeptide units -Gly-3(S)Hyp-4(R)Hyp- forms a trimer in aqueous solution The triple-helical nature of Gly-3(S)Hyp-4(R)Hyp is also supported by a previously determined crystal structure [29] In our previous paper [33], we used the 3(S)-Hyp commercially available from Fluka (Buchs, Switzerland) Amino acid analysis and MALDI-TOF mass spectroscopy showed the expected molecular weight (2892 Da) However, this peptide did not form a triple helix We attempted to determine why the previously used peptide did not form a triple helix The source of the 3(S)Hyp from Fluka that we used previously was hydrolyzed bovine collagen No information about the preparation of the commercial product is available from the company, and the product is not available in the USA 3(S)-hydroxyproline is known to degrade faster and isomerize to 3(R)Hyp more easily than 4(R)Hyp to 4(S)Hyp under acidic conditions [14] Therefore, hydrolysis of collagen could lead to the isomerization of 3(S)Hyp We used the method of Bellon et al [14] involving labeling with 4-chloro-7nitro-2,1,3-benzoxadiazole labeling and also labeling with 4-fluoro-7-nitrobenzofurazan to detect potential isomers of 3(S)Hyp, such as 3(R)Hyp and the d-isomer Unfortunately, the same batch of product that we used previously was no longer available and we did not have enough original peptide left for this analysis We could not detect a significant amount of 3(R)Hyp by thin-layer chromatography using a different batch of 3(S)Hyp from Fluka The peptide acetyl-(Gly3(S)Hyp-4(R)Hyp)10-NH2 has thirty 3(S)Hyp residues in the triple-helical structure If we assume that the presence of one incorrect 3(S)Hyp in the middle six tripeptide units causes the inability to form a triple helix, a 10% incorrect isomer content in the 3(S)Hyp preparation would mean that only 15% of the peptide could form a triple helix We assume that the 3(S)Hyp batch from Fluka that we used for the first preparation of the peptide contained a significant amount of isomerized 3(S)Hyp, but we not have enough peptide left to verify this hypothesis However, the results obtained by others [31] are consistent with this assumption The transition temperature (Tm) of peptide Ac-(Gly3(S)Hyp-4(R)Hyp)10-NH2 was determined by CD in H2O at 235 nm at a concentration of mm peptide with a heating rate of 7.5 °CỈh)1 (Fig 3A) The Tm of the peptide is 79.7 °C Under the same conditions, the Tm is a little higher than that of peptide Ac-(Gly-Pro4(R)Hyp)10-NH2 (76.1 °C) and very close to that of FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS K Mizuno et al 3-hydroxyproline in the collagen triple helix Table Thermodynamic values for the thermal transitions of Ac-(Gly-Xaa-Yaa)l0-NH2 peptides °, standard state for enthalpy and entropy change DSo DH° (JỈ°C)1Ỉ )1 (kJỈmol mol)1 Tm trimer) trimer) (°C) Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 Heating Cooling Ac-(Gly-Pro-4(R)Hyp)10-NH2 Heating Cooling Ac-(Gly-4(R)Hyp-4(R)Hyp)10-NH2 Heating Cooling (KJ·K–1·mol–1) Temperature (°C) Temperature (°C) Fig (A) Thermal transition curves of collagen-like peptides The peptides were measured in water, and the CD signal was monitored at 235 nm as a function of temperature The peptide concentration was mM and the temperature scanning rate was 7.5 °CỈh)1 Results are shown for Ac-(Gly-3(S)Hyp-4(R) Hyp)10-NH2 (square), Ac-(Gly-Pro-4(R)-Hyp)10-NH2 (upwards triangle) and Ac-(Gly-4(R)-Hyp-4(R)-Hyp)10-NH2 (circle) Both heating and cooling scans are shown The open symbols indicate heating scans, and the filled symbols indicate cooling scans (B) Differential scanning calorimetry of collagen-like peptides The peptides were dissolved in water, and scanned at 7.5 °CỈh)1 Results are shown for Ac-(Gly3(S)Hyp-4(R) Hyp)10-NH2 (solid line), Ac-(Gly-Pro-4(R)-Hyp)10-NH2 (dashed line) and Ac-(Gly-4(R)-Hyp-4(R)-Hyp)10-NH2 (dotted line) Both heating scans (positive values) and cooling scans (negative values) are shown The rate of temperature change is 7.5 °CỈh)1 in both directions The data for peptides Ac-(Gly-Pro-4(R) Hyp)10-NH2 and Ac-(Gly-4(R)Hyp-4(R)-Hyp)10-NH2 are from a previous study [34] and are included as a reference peptide Ac-(Gly-4(R)Hyp-4(R)Hyp)10-NH2 (80.5 °C) [34] The transition curve of peptide Ac-(Gly-3(S)Hyp4(R)Hyp)10-NH2 was not as sharp as that for Ac-(Gly- )207 )220 )337 )323 )169 )168 )482 )520 )858 )823 )385 )371 80.2 79.3 75.7 74.3 81.8 79.8 Pro-4(R)Hyp)10-NH2 In order to analyze the thermodynamic properties, the peptides were analyzed by DSC in water (Fig 3B) Peptide Ac-(Gly-3(S)Hyp4(R)Hyp)10-NH2 has a smaller transition enthalpy than peptide Ac-(Gly-Pro-4(R)Hyp)10-NH2, but a slightly larger transition enthalpy than Ac-(Gly-4(R)Hyp4(R)Hyp)10-NH2 [34] The transition enthalpies and entropies are summarized in Table Figure shows the proton NMR spectra of Ac-(Gly-4(R)Hyp-4(R)Hyp)10-NH2, Ac-(Gly-Pro-4(R) Hyp)10-NH2 and Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 In each of these three spectra, the large line-widths and the strong negative NOE cross-peaks are indicative of strong triple-helix formation Also, the resonances at approximately 3.1–3.4 p.p.m are markers for triple-helix formation as noted previously [34,35] This is further illustrated by its loss at high temperatures, shown on the left of Fig The fact that the line-widths are even greater in the Ac-(Gly-3(S)Hyp4(R)Hyp)10-NH2 spectrum may indicate a different degree of internal motions available to this species compared with the others Nevertheless, all of the spectra are characteristic of collagen triple helices Refolding of peptide H-(Pro-4(R)Hyp-Gly)10-OH is two orders of magnitude faster than that of peptide H-(Pro-Pro-Gly)10-OH [36] In order to assess the effect of 3(S)Hyp in the refolding kinetics, the peptides H-(Gly-3(S)Hyp-4(R)Hyp)9-OH, H-(Gly-Pro-4(R)Hyp)9OH and H-(Gly-4(R)Hyp-4(R)Hyp)9-OH were analyzed Refolding was monitored by CD at 225 nm in a concentration range from 2.7 · 10)2 mm to 1.0 mm at 10 °C The simple apparent initial reaction order [36] was calculated as shown below:   dẵH ẳ kẵC n dt tẳ0 where [C]0 is the initial peptide concentration, [H] is the concentration of triple-helical molecules, k is the rate constant, t is time, and n is the reaction order FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS 5833 3-hydroxyproline in the collagen triple helix K Mizuno et al Fig 1H-NMR spectra of collagen-like peptides Right: 2D NOESY data set for (A) Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2, (B) Ac-(GlyPro-4(R)Hyp)10-NH2 and (C) Ac-(Gly-4(R)Hyp4(R)Hyp)10-NH2, respectively Left: Variable temperature 1H-NMR spectra for Ac-(Gly3(S)Hyp-4(R)Hyp)10-NH2 at 75, 85, 95, 100 and 105 °C (from the bottom) Loss of the 3.2 p.p.m peak at high temperatures is consistent with the concomitant loss of triplehelix content Unless otherwise stated, the spectra were recorded at 30 °C, 10 mM concentration, and the NOESY mixing time was 60 ms The 2D NOESY data sets were processed using 60° phase-shifted sine bells before Fourier transformation, and the 1D spectra were treated with a Hz line-broadening factor As the fraction folded, F, is defined as the fraction of the peptide that forms a triple helix, F = 3[H] ⁄ [C]0, the equation   dF ẳ 3kẵC n1 dt t¼0 can be written as the logarithmic function   dF ẳ n 1ị logẵC ỵconst log dt t¼0 The apparent reaction order, n, can be obtained from the slope, (n ) 1), when the logarithm of the initial rate (dF ⁄ dt)t=0 is plotted on the y axis, and the logarithm of the total peptide concentration [C]0 is plotted on the x axis (Fig 5) The apparent reaction orders of peptides H-(Gly-Pro-4(R)Hyp)9-OH, H-(Gly-3(S)Hyp4(R)Hyp)9-OH and H-(Gly-4(R)Hyp-4(R)Hyp)9-OH were n = 1.3, 1.5 and 1.2, respectively These values are similar to the value n = 1.5 obtained for peptide H-(Pro-4(R)Hyp-Gly)10-OH at °C [36] Given the solubility of the peptides and the detection limits of the CD signals for analysis, it is virtually impossible to acquire data for higher or lower concentrations 5834 Within the measured concentration range, these three peptides refold much faster than peptide H-(Pro-ProGly)10-OH [36], implying that the 4(R)-hydroxyproline in the Yaa position contributes most to the folding rate of the model peptides, regardless of the Xaa position imino acid modification, i.e Pro, 3(S)Hyp or 4(R)Hyp The large difference in the rates of the H-(Pro-Pro-Gly)10-OH peptide and the other peptides at low concentrations indicates the importance of 4(R)Hyp in the nucleation process Future studies will be performed to study the mechanism of refolding of these model peptides in detail In order to evaluate the thermodynamic properties of 3(S)Hyp-containing collagen model peptides, a series of peptides with nine tripeptide units comprising -Gly-Pro4(R)Hyp- and -Gly-3(S)Hyp-4(R)Hyp- were synthesized, with the following sequences: H-(Gly-Pro-4(R)Hyp)9OH, H-(Gly-Pro-4(R)Hyp)4-Gly-3(S)Hyp-4(R)Hyp-(GlyPro-4(R)Hyp)4-OH, H-(Gly-Pro-4(R)Hyp)3-(Gly-3(S) Hyp-4(R)Hyp)2-(Gly-Pro-4(R)Hyp)4-OH, H-(Gly-Pro4(R)Hyp) -(Gly-3(S)Hyp-4(R)Hyp) -(Gly-Pro-4(R) Hyp)3-OH, H-(Gly-Pro-4(R)Hyp)1-(Gly-3(S)Hyp-4(R) Hyp)7-(Gly-Pro-4(R)Hyp)1-OH and H-(Gly-3(S)Hyp4(R)Hyp)9-OH The peptides were dissolved in NaCl ⁄ Pi FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS K Mizuno et al 3-hydroxyproline in the collagen triple helix 2.0 [θ]235 nm (deg cm2·dmol–1 × 10–3) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 60 70 80 Temperature (°C) Fig Refolding kinetics of peptides analyzed by CD Refolding of the collagen model peptides was monitored by CD at 225 nm The logarithm of the initial rate of triple-helix formation, log (dF ⁄ dt)t = 0, is plotted as a function of the logarithm of the total polypeptide chain concentration, log [C]0 The peptides H-(Gly-3(S)Hyp4(R)Hyp)9-OH (filled circle), H-(Gly-Pro-4(R)Hyp)9-OH (filled upwards triangle) and H-(Gly-4(R)Hyp-4(R)Hyp)9-OH (filled square) were measured at 10 °C The values for peptides H-(Pro-4(R)Hyp-Gly)10-OH (open upwards triangle) and H-(Pro-Pro-Gly)10-OH (open downwards triangle) measured at °C are from a previous study [36] and are included for comparison Fig Thermal transition curves of a series of 3(S)Hyp-containing peptides Peptides H-(Gly-Pro-4(R)Hyp)l-(Gly-3(S)Hyp-4(R)Hyp)m-(GlyPro-4(R)Hyp)n-OH, where (l, m, n) = (9, 0, 0), (4, 1, 4), (3, 2, 4), (3, 3, 3), (1, 7, 1) or (0, 9, 0), were analyzed by CD in NaCl ⁄ Pi at a peptide concentration of mM The CD signal was monitored at 235 nm with a heating rate of °CỈh)1 decrease in the transition enthalpies The numerical data are given in Table Discussion and analyzed by CD at 235 nm with a scanning rate of °CỈh)1 All peptides showed a cooperative transition, and little hysteresis was observed, as the heating and cooling curves almost overlapped under these experimental conditions As the number of 3(S)Hyp in the peptide increases, the slope of the transition becomes more shallow (Fig 6), indicative of a decrease in the transition enthalpy The set of peptides was also analyzed by DSC with a scanning rate of 0.1–2.0 °CỈmin)1 More than four repeating cycles of heating and cooling scans yielded overlapping curves, indicating that folding and unfolding is a reversible reaction under these conditions The transition temperatures of the heating and cooling scans were different with different scanning rates, but the scanning rate had no effect on the transition enthalpy for any peptide Figure shows the excess heat capacity of the peptides as a function of temperature Replacement of Pro by 3(S)Hyp decreases the transition enthalpy The first replacement of Pro by 3(S)Hyp in the middle of the triple helix has a large effect on the transition enthalpy Adding another also shows a further significant decrease of the transition enthalpy Further additions only lead to a minor Our new experimental data indicate that a Pro to 3(S)Hyp modification in the Xaa position of -GlyXaa-Yaa- collagen-like peptides increases the stability of the triple-helical structure by a small margin Insertion of 3(S)Hyp in the context of the nine tripeptide units increases the Tm of the peptides by approximately 0.5 °C per single replacement of -GlyPro-4(R)Hyp- by -Gly-3(S)Hyp-4(R)Hyp- Previously, we reported the crystal structure of the triple helix of peptide H(Gly-Pro-4(R)Hyp)3-(Gly-3(S)Hyp-4(R)Hyp)2(Gly-Pro-4(R)Hyp)4-OH [29] This structure is almost identical to the structure of other triple-helical (GlyPro-Pro)n or (Gly-Pro-4(R)Hyp)n peptides The height per tripeptide unit and the ⁄ symmetry were similar to those of other collagen peptides with imino acids in both the Xaa and Yaa positions [32] However, our previous analysis of the stability of Ac-(Gly-3(S)Hyp4(R)Hyp)10-NH2 seems inconsistent with this structure We therefore determined whether the presence of acetyl and amide groups in this peptide prevented triple-helical folding Jenkins et al [31] analyzed the host-guest peptide (Pro-4(R)Hyp-Gly)3-3(S)Hyp-4(R)Hyp-Gly-(Pro-4(R) Hyp-Gly)3-OH, and reported that the Pro to 3(S)Hyp FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS 5835 3-hydroxyproline in the collagen triple helix K Mizuno et al Table Thermodynamic values for the thermal transitions of H-(Gly-Pro-4(R)Hyp)l-(Gly-3(S)Hyp-4(R)Hyp)m-(Gly-Pro-4(R)Hyp)n-OH peptides Number of 3(S) Hyp per peptide DH° (kJỈmol)1 trimer) DS° (JỈ°C)1Ỉmol)1 trimer) Tm (°C)a )321 )224 )177 )170 )158 )156 )881 )581 )439 )418 )373 )370 52.0 52.8 51.0 52.6 56.2 55.3 a Tm (°C) ΔS (J·K–1·mole–1 trimer) Temperature (°C) modification lowered the Tm of the peptide by 3.3 °C When 3(S)fluoroproline was incorporated, a further decrease in the Tm was found [30] As the pKa of the carboxyl group of 3(S)Hyp is lower than that of Pro, these authors suggested that the hydrogen bond 5836 Data from heating scans at a scanning rate of 0.5 °CỈmin)1 between the amide group of Gly and the carbonyl group of 3(S)Hyp might be weaker than the hydrogen bond between the amide group of Gly and the carbonyl group of Pro They also analyzed the crystal structure of 3(S)Hyp-derived N-(13C2-acetyl)-3(S)-hydroxy-l-proline methyl ester, and the structure of the pyrrolidine ring is different from that of the N-(13C2-acetyl)-4(R)hydroxy-l-proline methyl ester We are not sure whether the lower Tm found in the host-guest peptide in their study is due to contamination or differences in the methods of observing the CD transition Our DSC analysis showed that peptide Ac-(Gly-3(S)Hyp4(R)Hyp)10-NH2 has a smaller transition enthalpy than peptide Ac-(Gly-Pro-4(R)Hyp)10-NH2 (Fig 3B) The smaller DH observed may be explained in several ways One is that the carbonyl group of 3(S)Hyp in the Xaa position is a weak hydrogen bond acceptor, as suggested by previous experimental data [31], because the carboxyl pKa value for 3(S)Hyp (1.62) is lower than that for Pro (1.92) The hydrogen bond between the amide of Gly and the carbonyl group of the residues in the Xaa position is the only direct inter-chain hydrogen bond in the triple helix Another explanation is that there is probably a difference in hydration of the unfolded chains The peptide with 3(S)Hyp could be more hydrated than the peptide with Pro in single chains, which would cause a decrease in the transition enthalpy and a decrease in the entropy of solvent water Fig Differential scanning calorimetry of a series of 3(S)Hyp-containing peptides (A) Peptides H-(Gly-Pro-4(R)Hyp)l-(Gly-3(S)Hyp4(R)Hyp)m-(Gly-Pro-4(R)Hyp)n-OH, where (l, m, n) = (9, 0, 0), (4, 1, 4), (3, 2, 4), (3, 3, 3), (1, 7, 1) or (0, 9, 0), were analyzed by DSC at mM peptide concentration in NaCl ⁄ Pi The excess heat capacity is shown as a function of temperature with a scanning rate of 0.5 °CỈmin)1 (B) Transition enthalpy per mole trimer (left axis) and the transition entropy (right axis) as a function of the number of 3(S)Hyp residues per chain (C) Transition temperatures (Tm) as a function of the number of 3(S)Hyp residues per chain, fitted using linear regression FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS K Mizuno et al molecules Kawahara et al [37] hypothesized that the difference in the degree of hydration explains the stability of the triple-helical structure of peptide H-(4(R)Hyp-4(R)Hyp-Gly)10-OH Recently, we analyzed the density of peptides with the repeated sequence (Gly4(R)Hyp-4(R)Hyp)n and (Gly-Pro-4(R)Hyp)n (n = and 9) over a wide range of temperature, and also analyzed the solution structure of these peptides by small-angle X-ray scattering (SAXS) [38] Our data indicate that at high temperatures, i.e unfolded chains, the peptides (Gly-4(R)Hyp-4(R)Hyp)9 and (Gly-Pro4(R)Hyp)9 have no significant structural differences Based on partial specific volume measurements, it is suggested that the hydration number for the peptide (Gly-Pro-4(R)Hyp)9 increases with formation of the triple-helix whereas that for the peptide (Gly-4(R)Hyp4(R)Hyp)9 decreases It is possible that peptides with 3(S)Hyp in the Xaa position also have these properties, and therefore a smaller transition enthalpy The change in the transition enthalpy in the series of host-guest 27-residue peptides was greatest for the first replacement of Pro by 3(S)Hyp The effect of additional substitutions of Pro by 3(S)Hyp was smaller than for the first substitution (Fig 7) Two junctions between Gly-Pro-4(R)Hyp and Gly-3(S)Hyp-4(R)Hyp are introduced by the first addition of a 3(S)Hyp residue, and this number remains constant upon addition of further 3(S)Hyp residues Therefore, the first introduction of a 3(S)Hyp residue probably changes the cooperativity and thermodynamic values more strongly than further additions We can rule out the possibility that absence of the 3(S)Hyp residue in type I collagen affects the stability of the triple helix, and stability or lack thereof is not the reason for the phenotypes observed when 3(S)Hyp is missing Mutations in P3H1 cause a form of lethal osteogenesis imperfecta [39], and knockout of the CRTAP gene encoding cartilage-associated protein in mouse causes a severe osteogenesis imperfecta-like phenotype [40] The proline at position 986 was not hydroxylated in the CRTAP null mice, as analyzed by mass spectroscopy However, it is not clear how the absence of 3(S)Hyp residues in type I collagen can cause these phenotypes We have ruled out stability as a factor It is much more likely that 3(S)Hyp takes part in protein–collagen interactions required for bone formation 3-hydroxylation of the proline at position 986 in the a1 chain of type I collagen involves the protein complex P3H1 ⁄ CRTAP ⁄ cyclophilin B [13,40] All functions of this complex in the rough endoplasmic reticulum need to be considered when analyzing the observed phenotypes Is the phenotype only due to absence of the 3-hydroxyproline in the collagen triple helix single 3(S)Hyp in the a1 chains of type I collagen or are other functions impaired as a result of mutations in the molecules of the complex? It may well be that 3(S)Hyp is important for bone mineralization, but other factors cannot be ruled out Bone protein and mineral interactions with type I collagen need to be further characterized to identify protein–collagen interactions that are affected by the lack of 3(S)Hyp in type I collagen Experimental procedures Peptide synthesis Peptides were synthesized using an ABI 433A synthesizer (Applied Biosystems, Foster City, CA, USA) Couplings were performed using Fmoc-PAL-PEG-PS resin (0.16 mmolỈg)1) (Applied Biosystems) for the C-terminal amide-capped peptides, or O-t-butyl-l-trans-4-hydroxyproline-2-chlorotrityl resin (0.48 meqỈg)1) (AnaSpec, San Jose, CA, USA) for peptides with 4(R)Hyp at the C-terminal end Fmoc-amino acids Fmoc-Gly-OH and Fmoc-Pro-OH were purchased from Applied Biosystems, Fmoc-4(R) Hyp(tBu)-OH was purchased from Novabiochem (EMD Biosciences Inc., San Diego, CA, USA), and acetyl glycine was purchased from Bachem (Torrance, CA, USA) Commercially available Fmoc-3(S)-hydroxyproline (AnaSpec) was used without any further purification HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (Applied Biosystems) (4.0 eq) and di-isopropylethylamine were used as the coupling reagents for the Fmoc solid-phase peptide synthesis The peptides were cleaved from the resin using Reagent R (trifluoroacetic acid ⁄ thioanisole ⁄ 1,2-ethanedithiol ⁄ anisole, 90 : : : 2) at room temperature for h Peptides were isolated by precipitation from the cleavage cocktail with diethyl ether at °C, dissolved in 0.1% trifluoroacetic acid and purified by preparative HPLC using a VydacÒ ˚ 218TP101550 C18 column (5 lm internal diameter, 300 A pore size, 50 · 250 mm) and a 218TP15202503 guard column (W.R Grace & Co., Columbia, MD, USA) with a flow rate of 36 mLỈmin)1 and elution with a 0–50% acetonitrile gradient in 0.1% trifluoroacetic acid All peptides were characterized by electrospray ⁄ quadrupole ⁄ time-of flight mass spectrometry (Q-tof micro, Waters Corp., Milford, MA, USA), and amino acid analysis The Ac-(Gly-Pro-3(S)Hyp)10-NH2 peptide was also characterized by MALDI-TOF at the Stanford Protein and Nucleic Acid facility (Stanford, CA, USA) The Ac-(Gly-3(S)Hyp4(R)Hyp)10-NH2 peptide was analyzed by MALDI-TOF at the Department of Dentistry, Oregon Health and Science University (Portland, OR) The peptides were stored at )20 °C before preparing stock solutions The stock solutions for analysis were stored at °C FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS 5837 3-hydroxyproline in the collagen triple helix K Mizuno et al Circular dichroism CD spectra were recorded on an Aviv 202 spectropolarimeter (Aviv Biomedical Inc., Lakewood, NJ, USA) using a Peltier thermostatted cell holder and a mm path-length rectangular quartz cell (Starna Cells Inc., Atascadero, CA, USA) Peptide concentrations were determined by amino acid analysis (L-8800A amino acid analyzer; Hitachi High Technologies America Inc., San Jose, CA, USA) The wavelength spectra represent the mean of at least 10 scans, with 0.1 nm resolution of one second averaged data The temperature scanning experiments were run at 0.1 °CỈmin)1 For determination of refolding kinetics, the peptides were denatured at 75 °C for 10 min, and then rapidly diluted at ratios from : to : 64 with solvent cooled on ice After rapid mixing, the sample solution was immediately put into the mm path-length rectangular quartz cuvette cell in the CD spectrometer holder pre-incubated at 10 °C The ellipticity at 225 nm was monitored as a function of time The fraction of folded peptide (F) is defined as F¼ hobs À hDN hN À hDN where hobs, hDN and hN represent the observed, monomeric and triple-helical peptide ellipticity, respectively hN was measured directly at the temperature used for refolding (10 °C) hDN was determined by linear extrapolation of the straight line measured under denatured conditions between 75 and 85 °C Analytical ultracentrifugation Sedimentation-equilibrium analysis was performed using a Beckman Coulter ProteomeLabÔ model XL-A analytical ultracentrifuge (Beckman Coulter, Inc., Fullerton, CA, USA) The An-60 Ti rotor was used together with 12 mm Epon centerpiece double-sector cells with quartz windows The peptides were analyzed in 20 mm phosphate buffer, pH 7.2, containing 150 mm NaCl, unless otherwise indicated The peptide concentrations were adjusted from 0.02 to 0.1 mgỈmL)1 Sedimentation equilibrium measurements were performed at temperatures of or 25 °C The analysis was performed using scientist software (Micromath, St Louis, MO, USA) with the assumption that there is a single molecular species in the solution A partial specific volume of 0.67 mLỈg)1 was used for all calculations Differential scanning calorimetry Differential scanning calorimetry was performed using a Nano II model 6100 differential scanning calorimeter (Calorimetry Science Corporation) with 0.299 mL capillary cells A stock sample solution in water was prepared and adjusted to the required peptide concentration in the sample solution The peptide sample in NaCl ⁄ Pi was 5838 de-gassed before analysis The heating and cooling scans of the peptides used in this experiment were all reproducible in several repeat scans The heating and cooling rates ranged between 0.1 and 2.0 °CỈmin)1 The data were analyzed using cpcalc software (Calorimetry Science Corporation, Lindon, UT, USA) The DCp values for the peptides analyzed in this paper were assumed to be zero All the heat-capacity curves were fitted using the polynomial baseline fit The concentration of the peptide was determined by amino acid analysis NMR spectroscopy NMR spectra were recorded on a Bruker AMX-400 spectrometer operating at 400.14 MHz (Bruker, Madison, WI, USA) The 90° pulse width was ls, and a low-power s presaturation pulse was applied to suppress the H2O (HOD) resonance The spectra were recorded as 16 384 points for the 1D spectra, and as 1024 · 512 data point sets for the 2D spectra NOESY data were collected with time proportional phase increment in the indirect dimension, at mixing times between 30 and 120 ms, and with a total recording time of approximately 10 h TOCSY data were collected with various mixing times, ranging from 30 to 90 ms The data were processed using swan-mr to 1024 · 1024 real data sets after application of a 60° phaseshifted sin2 function and Fourier transformation for the 2D spectra; baselines were straightened using polynomials as required Spectra were referenced to p.p.m via internal 2,2-dimethylsilapentane-5-sulfonate or via the water resonance (4.71 p.p.m at 30 °C) Final visualization and analyses of the 2D data sets were performed using nmrview [41] or swan-mr [42] Acknowledgements This work was supported by a grant from Shriners Hospital for Children (Portland, OR, USA) The authors thank Eric A Steel and Jessica L Hacker for expert technical assistance and Dr B Kerry Maddox for amino acid analyses References Bachinger HP & Engel J (2005) The thermodynamics ă and kinetics of collagen folding In Protein Folding Handbook (Buchner J & Kiefhaber T, eds), pp 1059– 1110 Wiley, New York, NY Okuyama K, Xu X, Iguchi M & Noguchi K (2006) Revision of collagen molecular structure Biopolymers 84, 181–191 Myllyharju J & Kivirikko KI (2004) Collagens, modifying enzymes and their mutations in humans, flies and worms Trends Genet 20, 33–43 FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS K Mizuno et al Wolff JS III, Logan MA & Ogle JD (1966) Evidence for the acceptance of 3-hydroxyproline as a new imino acid constituent of proteins Fed Proc 25, 862–863 Wolff JS III, Ogle JD & Logan MA (1966) Studies on 3-methoxyproline and 3-hydroxyproline J Biol Chem 241, 1300–1307 Gaill F, Mann K, Wiedemann H, Engel J & Timpl R (1995) Structural comparison of cuticle and interstitial collagens from annelids living in shallow sea-water and at deep-sea hydrothermal vents J Mol Biol 246, 284–294 Kimura S & Matsuura F (1974) The chain compositions of several invertebrate collagens J Biochem 75, 1231– 1240 Kimura S, Kamimura T, Takema Y & Kubota M (1981) Lower vertebrate collagen Evidence for type Ilike collagen in the skin of lamprey and shark Biochim Biophys Acta 669, 251–257 Takema Y & Kimura S (1982) Two genetically distinct molecular species of octopus muscle collagen Biochim Biophys Acta 706, 123–128 10 Miura S & Kimura S (1985) Jellyfish mesogloea collagen Characterization of molecules as a1a2a3 heterotrimers J Biol Chem 260, 15352–15356 11 Risteli J, Tryggvason K & Kivirikko KI (1977) Prolyl 3-hydroxylase: partial characterization of the enzyme from rat kidney cortex Eur J Biochem 73, 485–492 12 Risteli J, Tryggvason K & Kivirikko KI (1978) A rapid assay for prolyl 3-hydroxylase activity Anal Biochem 84, 423–431 13 Vranka JA, Sakai LY & Bachinger HP (2004) Prolyl ă 3-hydroxylase 1, enzyme characterization and identication of a novel family of enzymes J Biol Chem 279, 23615–23621 14 Bellon G, Berg R, Chastang F, Malgras A & Borel JP (1984) Separation and evaluation of the cis and trans isomers of hydroxyprolines: effect of hydrolysis on the epimerization Anal Biochem 137, 151–155 15 Marquardt H, Wilson CB & Dixon FJ (1973) Human glomerular basement membrane Selective solubilization with chaotropes and chemical and immunologic characterization of its components Biochemistry 12, 3260– 3266 16 Ohno M, Riquetti P & Hudson BG (1975) Bovine renal glomerular basement membrane Isolation and characterization of a glycoprotein component J Biol Chem 250, 7780–7787 17 West TW, Fox JW, Jodlowski M, Freytag JW & Hudson BG (1980) Bovine glomerular basement membrane Properties of the collagenous domain J Biol Chem 255, 10451–10459 18 Click EM & Bornstein P (1970) Isolation and characterization of the cyanogen bromide peptides from the a1 and a2 chains of human skin collagen Biochemistry 9, 4699–4706 3-hydroxyproline in the collagen triple helix 19 Lane JM & Miller EJ (1969) Isolation and characterization of the peptides derived from the a2 chain of chick bone collagen after cyanogen bromide cleavage Biochemistry 8, 2134–2139 20 Miller EJ, Martin GR, Piez KA & Powers MJ (1967) Characterization of chick bone collagen and compositional changes associated with maturation J Biol Chem 242, 5481–5489 21 Fietzek PP, Rexrodt FW, Wendt P, Stark M & Kuhn ă K (1972) The covalent structure of collagen Aminoacid sequence of peptide a1-CB6-C2 Eur J Biochem 30, 163–168 22 Kielty C & Grant M (2002) The collagen family: structure, assembly and organisation in the extracellular matrix In Connective Tissue and its Heritable Diseases, 2nd edn (Royce PM & Steinmann B, eds), pp 159–221 Wiley-Liss, New York, NY 23 Raines RT (2006) 2005 Emil Thomas Kaiser Award Protein Sci 15, 1219–1225 24 Sakakibara S, Inouye K, Shudo K, Kishida Y, Kobayashi Y & Prockop DJ (1973) Synthesis of (Pro-HypGly)n of defined molecular weights Evidence for the stabilization of collagen triple helix by hydroxypyroline Biochim Biophys Acta 303, 198–202 25 Engel J, Chen HT, Prockop DJ & Klump H (1977) The triple helix in equilibrium with 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 16, 601–622 26 O’Hagan D & Rzeoa HS (1997) Some influences of fluorine in bioorganic chemistry Chem Commun 7, 645– 652 27 Mizuno K, Hayashi T & Bachinger HP (2003) Hydroxă ylation-induced stabilization of the collagen triple helix Further characterization of peptides with 4(R)-hydroxyproline in the Xaa position J Biol Chem 278, 32373– 32379 28 Bann JG & Bachinger HP (2000) Glycosylaă tion hydroxylation-induced stabilization of the collagen triple helix 4-trans-hydroxyproline in the Xaa position can stabilize the triple helix J Biol Chem 275, 24466–24469 29 Schumacher MA, Mizuno K & Bachinger HP (2006) ¨ The crystal structure of a collagen-like polypeptide with 3(S)-hydroxyproline residues in the Xaa position forms a standard ⁄ collagen triple helix J Biol Chem 281, 27566–27574 30 Hodges JA & Raines RT (2005) Stereoelectronic and steric effects in the collagen triple helix: toward a code for strand association J Am Chem Soc 127, 15923– 15932 31 Jenkins CL, Bretscher LE, Guzei IA & Raines RT (2003) Effect of 3-hydroxyproline residues on collagen stability J Am Chem Soc 125, 6422–6427 FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS 5839 3-hydroxyproline in the collagen triple helix K Mizuno et al 32 Okuyama K, Wu G, Jiravanichanun N, Hongo C & Noguchi K (2006) Helical twists of collagen model peptides Biopolymers 84, 421–432 33 Mizuno K, Hayashi T, Peyton DH & Bachinger HP ă (2004) The peptides acetyl-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 and acetyl-(Gly-Pro-3(S)Hyp)10-NH2 not form a collagen triple helix J Biol Chem 279, 282–287 34 Mizuno K, Hayashi T, Peyton DH & Bachinger HP ă (2004) Hydroxylation-induced stabilization of the collagen triple helix Acetyl-(glycyl-4(R)-hydroxyprolyl-4(R)hydroxyprolyl)10-NH2 forms a highly stable triple helix J Biol Chem 279, 38072–38078 35 Feng Y, Melacini G, Taulane JP & Goodman M (1996) Acetyl-terminated and template-assembled collagenbased polypeptides composed of Gly-Pro-Hyp sequences Synthesis and conformational analysis by circular dichroism, ultraviolet absorbance, and optical rotation J Am Chem Soc 118, 10351–10358 36 Boudko S, Frank S, Kammerer RA, Stetefeld J, Schulthess T, Landwehr R, Lustig A, Bachinger HP & ă Engel J (2002) Nucleation and propagation of the collagen triple helix in single-chain and trimerized peptides: transition from third to first order kinetics J Mol Biol 317, 459–470 37 Kawahara K, Nishi Y, Nakamura S, Uchiyama S, Nishiuchi Y, Nakazawa T, Ohkubo T & Kobayashi Y (2005) Effect of hydration on the stability of the collagen-like triple-helical structure of [4(R)-hydroxyprolyl-4(R)-hydroxyprolylglycine]10 Biochemistry 44, 15812–15822 38 Terao K, Mizuno K, Murashima M, Kita Y, Hongo C, Okuyama K, Norisuye T & Bachinger HP (2008) Chain ¨ dimensions and hydration behavior of collagen model peptides in aqueous solution: [glycyl-4(R)-hydroxy- 5840 39 40 41 42 prolyl-4(R)-hydroxyproline]n, [glycylprolyl-4(R)hydroxyproline]n, and some related model peptides Macromolecules, 41, 7203–7210 Cabral WA, Chang W, Barnes AM, Weis M, Scott MA, Leikin S, Makareeva E, Kuznetsova NV, Rosenbaum KN, Tifft CJ et al (2007) Prolyl 3-hydroxylase deficiency causes a recessive metabolic bone disorder resembling lethal ⁄ severe osteogenesis imperfecta Nat Genet 39, 359–365 Morello R, Bertin TK, Chen Y, Hicks J, Tonachini L, Monticone M, Castagnola P, Rauch F, Glorieux FH, Vranka J et al (2006) CRTAP is required for prolyl 3-hydroxylation and mutations cause recessive osteogenesis imperfecta Cell 127, 291–304 Johnson BA & Blevins RA (1994) A computer program for the visualization and analysis of NMR data J Biomol NMR 4, 603–614 Balacco G (2000) SwaN-MR: from infancy to maturity Mol Biol Today 1, 23–38 Supporting information The following supplementary material is available: Fig S1 Sedimentation equilibrium analysis of collagen-like peptides containing 3(S)Hyp This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary material supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 5830–5840 ª 2008 The Authors Journal compilation ª 2008 FEBS ... modification in the Xaa position of -GlyXaa-Yaa- collagen- like peptides increases the stability of the triple-helical structure by a small margin Insertion of 3(S)Hyp in the context of the nine... and the other peptides at low concentrations indicates the importance of 4(R)Hyp in the nucleation process Future studies will be performed to study the mechanism of refolding of these model peptides. .. position contributes most to the folding rate of the model peptides, regardless of the Xaa position imino acid modification, i.e Pro, 3(S)Hyp or 4(R)Hyp The large difference in the rates of the