- Email: [email protected]
Stability of type I collagen CNBr peptide trimers1
J. Mol. Biol. (1997) 269, 488±493
COMMUNICATION
Stability of Type I Collagen CNBr Peptide Trimers Antonio Rossi, Giuseppe Zanaboni, Giuseppe Cetta and Ruggero Tenni* Dipartimento di Biochimica ``Alessandro Castellani'', via Taramelli 3b, University of Pavia, 27100 Pavia, Italy
As indices of triple helix stability of type I collagen CNBr peptide homotrimers, G for monomer-trimer transitions and melting temperatures were obtained from circular dichroism measurements at increasing temperatures. The data were compared with the stability of the parent native molecule. Peptides were found to have a lower stability than the whole collagen molecule. The general implication is that the coordinated water molecules play a key role in determining collagen triple helical stability and high cooperativity at melting. Other factors (monomer stability, ionic and hydrophobic factors, variations of composition, speci®c sequences) could also contribute towards peptide stability; these factors could explain the data obtained in the case of peptide a1(I) CB3. # 1997 Academic Press Limited
*Corresponding author
Keywords: collagen; collagen peptides; triple helix stability; cooperative melting
The stability of the collagen triple helix has been the subject of extensive work. Privalov (1982) has reviewed and discussed this work in terms of the thermodynamics of collagen structure stabilization, its correlation with the chemical characteristics of collagen structure and the participation of water in its stabilization. Hydroxyproline and the extended network of coordinated water molecules are major contributors to collagen stability. Recently, the pivotal role of the Hyp hydroxyl group for the regular water networks has been clearly demonstrated after determination of the molecular and the crystal structures of the peptide (Pro-Hyp-Gly)4-Pro-HypAla-(Pro-Hyp-Gly)5 (Bella et al., 1994, 1995). Other factors involved in collagen structure stabilization have also been reported; these were mainly based on computer modeling: side-chain interactions by Vitagliano et al. (1993); electrostatic interactions (Venugopal et al., 1994; charged tripoles: Katz & David, 1992); hydrophobic interactions (Jones & Miller, 1991; interchain proline-proline contacts: Bhatnagar et al., 1988). For a review on these aspects see VaÂsquez et al. (1994). We recently puri®ed CNBr peptides covering more than 96% of a1(I) and a2(I) chains of acid-soluble type I collagen from calf skin and analysed the molecular species peptides can form, their conAbbreviations used: Hyp, hydroxyproline; CD, circular dichroism; tm, melting temperature. 0022±2836/97/240488±6 $25.00/0/mb971046
formation and thermodynamic and kinetic aspects of equilibria between species. Single-stranded molecules (monomers) of all peptides are able to form homotrimers with triple helical conformation; trimers of some peptides are able to auto-aggregate. The stability of trimers merits futher consideration, because a high fraction of peptide monomers fold into trimers, but full helicity is not reached, and because melting temperatures for peptides are lower than that obtained for native type I collagen molecules (Rossi et al., 1996). Only the largest peptide we analysed, a2(I) CB3,5, showed irreversible denaturation such as occurs for collagen. Using circular dichroism, the positive signal occurring at 221 nm, which is typical of native collagen and trimeric peptides in a triple-helical conformation, was found to increase very slowly with time for this peptide and we found variations after nine months of equilibration at 4 C (data not shown). Since Hyp content and total imino acid content differ for the peptides (ranging from 82 to 167 and 198 to 333 residues per 1000 residues, respectively), we analysed the thermodynamic aspects of peptide trimer stability, to investigate their contribution to the stability of the whole molecule. As reference, we used data cited in Privalov's work and data on type I collagen CNBr peptides (Saygin et al., 1978). We could not make any comparison with type I collagen homotrimer, [a1(I)]3, since thermodynamic data similar to ours have not been reported for this collagen type. # 1997 Academic Press Limited
Stability of Type I Collagen CNBr Peptide Trimers
489
Experimental data were obtained by following the decrease of the CD signal at 221 nm at increasing temperatures (for the procedure used, see Rossi et al., 1996). The denaturation of collagen is a slow process, as is the peptide trimer to monomer conversion (Rossi et al., 1996). The apparent melting curve is therefore more or less close to the equilibrium melting curve. Thus, we used low heating rates, as suggested by Privalov (1982), and comparable with those used in similar experiments by Long et al. (1993) and Venugopal et al. (1994). The (van't Hoff) enthalpy change we obtained for bovine type I collagen melting was 379 kcal molÿ1, quite similar to the value from calorimetric measurements reported for rat skin collagen (Privalov, 1982). Melting temperatures, van't Hoff (or effective) H , S and G were calculated using the procedure of Engel et al. (1977) by using their equations 11, 7, 5 and 4. The structure of the mathematical procedure (the set of formulas) for computing the parametric values is such that the parameters are not independent (H f(c, tm, F); S f(c, tm, H ); G f(H , S ), where c is total monomer concentration and F is the fraction of chains in helical state at each temperature tested). We also obtained data on the monomer drift, i.e. the variation of monomer CD signal with temperature. Stability of peptides Melting temperature and G of the monomertrimer transition are indices of peptide trimer stability. They were found to be correlated (r2 0.993 for all samples analysed; r2 0.994 for peptides from a1(I), with the exclusion of CB3) (data not shown). When normalized per unit length (per triplet), all thermodynamic parameters correlated with Hyp content and, better still, with total imino acid content (Figures 1 and 2). It is evident from Figure 1 that enthalpic factors are the driving forces for the spontaneity of trimer formation, with a concomitant decrease of entropy. The peptide located in the middle of a1(I), CB3, behaves differently in that it shows high ``extra-stability''. This result is not artifactual as it was con®rmed in ®ve different determinations using three different preparations. Standard deviation was <10% of the mean for all thermodynamic parameters. Privalov (1982) assumed that the contribution made by Hyp towards collagen stability is due to two factors: a contribution identical to that of Pro, and a speci®c contribution for Hyp ability to act as the coordination centre of water molecules. The data concerning these contributions have been calculated for peptides and compared with Privalov's data for collagens (Table 1). The ordered water shell contributes towards enthalpic helical stability and greatly decreases entropy for both collagens and peptides. This special Hyp contribution is lower for peptides than for the whole molecule.
Figure 1. Plots for peptide trimer formation (monomertrimer transition) of the thermodynamic parameters, normalized per unit length (per triplet), versus % imino acid content and versus % Hyp content. The following peptides were analysed: a1(I) CB2 - CB8 - CB3 - CB7 CB6 and composite peptides CB2,4 - CB3,7 and CB3,8 (®lled circles, except CB3 (diamond)); a2(I) CB4 - CB3,5 (®lled squares). Open circle, acid-soluble type I collagen. Interpolating lines were for peptides from a1(I) only (continuous lines) or also for peptides from a2(I) (broken lines); r2 is indicated for each interpolation. The data for CB3 and collagen were excluded from all interpolations.
This ®nding can be explained by the presence of a less extended and/or ordered hydration shell. The crossover of all interpolating lines in Figures 1 and 2 with the value 0 of all parameters is near 20% total imino acid content and 10% Hyp content. Thus, a minimum of about 100 Hyp residues per 1000 residues and about 200 residues of Pro Hyp are required for trimer formation for the peptides we analysed. Peptides have a Hyp/ Pro ratio in the range 0.71 to 1.38 (0.81 to 1.18 for peptides from a1(I) only, those from a2(I) being at the extremes of the range). By assuming a ratio of 1:1, the spontaneity of trimer formation at 25 C, i.e. when G just turns negative, can be calculated
490
Stability of Type I Collagen CNBr Peptide Trimers
Figure 2. Plots of melting temperatures (calculated at the formal concentration of 1 mM) of peptide trimers and of the linear variation with temperature of CD signal at 221 nm for peptide monomers. Symbols are as in Figure 1.
from the equations given in Table 1. A minimum content of 200 residues of Hyp Pro per 1000 residues is required both for collagens and peptides. The most important ®nding is the similarity of the general Hyp contribution to G in the case of both peptides and collagens, in concomitance with
a lower water contribution to peptide stability, about 1/3.4 with respect to the value for collagen (see equations for G in Table 1). If we assume that, on average, peptide homotrimers share with their parent native molecule the same characteristic one interchain hydrogen bond and the same average Hyp content and total imino acid content, we can conclude with the more general view that the greatest contribution to the stability of the collagen triple helix is given by the extended and highly ordered hydration shell. This could also explain the very high degree of cooperativity of collagen melting. This last point raises the question of the number and size of cooperative blocks. Type I collagen melts with a very sharp transition and a very large enthalpy change, as measured by calorimetric studies. Calorimetric enthalphy is about 12 times greater than van't Hoff (or effective) enthalphy. Collagen melting was thus interpreted as an extremely cooperative process and the length of each cooperative block, calculated from the ratio calorimetric/van't Hoff enthalpy, is about 1000/12, i.e. about 80 residues per chain or about 250 residues per trimer (Privalov, 1982). Type II collagen has very similar characteristics with a cooperative block length of about 60 residues (BaÈchinger & Morris, 1990). Slightly different values for types I and II collagens are reported by Davis & BaÈchinger (1993), together with data for type III collagen. Other collagens of the ®brillar family show complex transitions (Morris et al., 1990). In this context, we compared our enthalpy data obtained by optical measurements (van't Hoff enthalpies) with calorimetric enthalpy data (Saygin et al., 1978). Unfortunately, the latter data concern only a very few of the peptides we considered (CB2, CB3, CB7 and CB8). We were also unable to ®nd calorimetric data for peptide CB6 that covers a
Table 1. Thermodynamic data of monomer-trimer transition for peptides versus data for collagen H (kcal molÿ1) ÿ 574 ÿ 8.56 ZHyp 930 ÿ 9.87 ZHyp ÿ 551 ÿ 0.74 ZP Hyp ÿ 7.35 ZHyp(w) 1077 ÿ 3.90 ZP Hyp ÿ 3.28 ZHyp(w) S (cal Kÿ1 molÿ1) ÿ 2630 1.43 ZP Hyp ÿ 23.4 ZHyp(w) 3264 ÿ 12.0 ZP Hyp ÿ 9.65 ZHyp(w) G (kcal molÿ1) 215 ÿ 0.43 ZP Hyp ÿ 1.34 ZHyp(w) 103 ÿ 0.32 ZP Hyp ÿ 0.39 ZHyp(w)
r2
Collagen (equation 4 in *) Peptides Collagen (from table V in *) Peptides
0.923 0.946 0.991
Collagen (equation 6 in *) Peptides
0.990
Collagen (equation 7 in *, modi®ed) Peptides (a)
0.996
Data for collagen are from Privalov (1982) (indicated in the Table with a *); data for peptides are from a1(I) only, with the exclusion of CB3. The values are computed for 1000 residues. When based on experimental data, the values which best ®t the equations >parameter a b ZP Hyp c ZHyp(w) are given; in this case, r2 for the linear correlation between experimental and calculated data is given. Z, residues/1000 residues of the imino acid(s) in superscript; Hyp(w), denotes the contribution of Hyp as the coordination centre of water molecules. (a) Due to the signi®cant correlation between G and tm, one could also obtain an estimation of tm from peptide imino acid content by the equation: tm,1000 ( C) ÿ 201 0.52 ZP Hyp 0.98 ZHyp(w) (r2 0.985) and then by: tm ( C) tm,1000 number of residues in the peptide trimer / 1000.
491
Stability of Type I Collagen CNBr Peptide Trimers
stable region at the C-terminal end of the a1(I) chain and for peptides from the a2(I) chain. Three results arise from this comparison: the length of the cooperative blocks we can calculate for peptides is of the same amplitude of those for collagen, the number of cooperative blocks is much lower for peptides, and peptides differ from each other: 1.3 cooperative blocks with 30 residues/block for peptide CB2, 2.0 cooperative blocks with 75 residues/block for CB3, 2.8 blocks with 95 residues/ block for CB7, 2.3 blocks with 120 residues/block for CB8. From these data we have the indication of a higher than average stability for the N-terminal peptide and for a central one (CB2 and CB3, respectively; CB6 probably shares this characteristic), and a lower stability for the other peptides. Furthermore, block length is not dependent on peptide length. For all peptides we can reasonably assume that the very same factor, i.e. the contribution of multiple weak bonds of Hyp/water shell, is a key factor not only towards maintaining stability but also for the modulation of the degree of cooperativity at melting. Such a factor is lower for peptides and therefore other stabilizing factors could become more evident for the stability of certain peptides, like CB3. Stability of CB3 Results of calorimetric measurements showed that CB3 did not present any extra-stability (see Table III and Figure 5 of Saygin et al., 1978). The
extra-stability we saw in our data is thus due to the cooperative block length. The result is therefore the unexpected ®nding of the presence of high triple helical stability or a lower than average block length not only at the terminal ends (BaÈchinger & Davis, 1991), due to their high Hyp and total imino acid content, but also in the central portion spanned by CB3 (residues 403 to 551 of the triple helix). This CB3 stability is not the result of its imino acid content (see Figure 1); so other factors involved have no longer a quantitatively marginal role. A ®rst clue to support the stability of CB3 comes from the calculated relative stability along type I collagen and a1(I) chain (see Figure 2 of BaÈchinger & Davis, 1991): the CB3 region has the highest mean stability. It has also the lowest dispersion around the mean and for this peptide, at variance from all others, no profound minima, i.e. no zones of very low relative stability, are present. Furthermore, the drift of monomer CD signal at increasing temperatures is close to zero for CB3 (Figure 2) and is compatible with its imino acid content. The monomer drift can be read as the variation of the secondary structure that monomers maintain for the presence of rigid imino acids. CB3 monomer is present in the most stable or disordered structure even at 25 C and, therefore, G is the difference between two states (monomer and trimer), the former being already at its energetic minimum. Charged tripoles arise from Gly-Xÿ-Y sequences. Positive and negative tripoles have an or-
Table 2. Amino acid composition of some selected collagen polypeptide chains (residues/1000 residues)
D E H K R Hyp S T Y C G N Q A F I L M P V W
Type I collagen
Whole
26.6 47.7 4.9 32.9 51.3 109.1 31.9 17.1
29.6 48.3 2.0 35.5 50.3 116.4 32.5 16.8
335.3 16.8 24.0 112.1
335.3 12.8 24.7 118.3
12.2 11.2 24.0 6.2 113.4 23.3
11.8 7.9 18.7 6.9 116.4 15.8
a1(I) Gap
Ov.
Whole
a1(I) CB3 Gap
Ov.
Whole
26.8 67.1
18.5 92.6
31.6 52.6
39.7 49.6 101.2 27.8 19.8
23.5 43.1 3.9 31.4 51.0 131.4 37.2 13.7
33.6 40.3 114.1 20.1
55.6 37.0 92.6
21.1 42.1 126.3 31.6
36.5 41.7 5.2 26.0 57.3 114.6 52.1 20.4
335.3 11.9 19.8 140.9 (49%) 6.0 6.0 9.9 6.0 119.0 17.9
335.3 13.7 29.4 96.1 (53%) 17.6 9.8 27.5 7.8 113.7 13.7
335.6 13.4 40.3 147.7
333.3 37.0 166.7 (33%) 18.5
336.8 21.1 42.1 136.8 (62%) 21.1
111.1 37.0
31.6 10.5 73.2 21.1
35.7 53.6
20.1 20.1 6.7 87.2 26.8
333.3 5.2 20.8 93.8 5.2 15.6 20.8 140.6 10.4
a1(I) CB6 Gap 39.2 68.6 29.4 58.8 88.2 39.2 29.4 333.3 19.6 127.5 (54%) 9.8 9.8 127.5 19.6
Ov. 33.3 11.1 11.1 22.2 55.6 144.4 66.7 11.1 333.3 11.1 22.2 55.6 (20%) 11.1 22.2 33.3 155.6
The composition was calculated from bovine a1(I) and a2(I) sequences, where known; otherwise, human sequences were used (Galloway, 1982; Phillips et al., 1992). Characters in italics indicate difference with a1(I); in bold indicate a difference between gap and overlap zone. The values in parenthesis are the percentage of alanine in X position. Positioning of residues in gap and overlap (Ov.) zones. CB3: gap, residues 403 to 456 (54 res. long); overlap, res. 457 to 551 (95 res.). CB6: gap, res. 823 to 924 (102 res.); overlap, res. 925 to 1014 (90 res.) (from Jones & Miller, 1991)
492 dered relative disposition along the triple helix and this contributes to triple helix chirality and to the azimuthal orientation of adjacent triple helices (Katz & David, 1992). They can also contribute to collagen and CB3 stability. Four out of 14 negative tripoles, and probably the same fraction of positive ones, fall into the CB3 region (Katz & David, 1992). Other, but unquanti®able, clues come from the composition of the peptides as a whole or of their portions that fall into gap and overlap zones of the ®brils (Table 2). This difference among the two zones is here brought in evidence because, by applying the equation in Table 1 that relates G with Hyp and total imino acid contents, it can be calculated that by far the greatest contribution to stability is given by the portions falling into the overlap zones. Several amino acids are differently represented in the two zones of a1(I) chain and of CB3 and CB6. We performed this calculation only for these two peptides because in the simplest way they start in the gap zone and end in the overlap one; other peptides have a more complex disposition in the two zones. Some amino acids, apart from Pro and Hyp, have a preferential X or Y position, but there are not signi®cant differences between the two zones for any amino acid, with the relevant exception of alanine. When isolated chains fold into trimers, hydrophobic amino acids have a decreased area in contact with the solvent, especially when they are in the Y position (Jones & Miller, 1991). Thus, it may be relevant that the CB3 gap zone is devoid of the largest amino acids of this class. Rather, the CB3 gap zone is particularly more rich in proline residues than the overlap zone is, differing from a1(I) and CB6, and in glutamic acid and lysine. However, a much more complex picture emerges if one takes into account the composition of the stretch in a2(I) corresponding to CB3 in a1(I). But this stretch is also poor in Hyp and total imino acid content (data not shown), and therefore the stability of the central region of type I collagen is not brought about exclusively by the presence of a2(I) chain.
Conclusive remarks Type I collagen CNBr peptides are able to form (homo)trimers. Here we have presented thermodynamic data concerning the stability of peptide trimers in comparison with the stability of the whole molecule. We conclude that: (1) peptides have a lower stability than the parent molecule, mainly because of a less extended and/or structured water shell; (2) this implies that the key feature involved in collagen triple helical stability and in the very high cooperativity at melting is the presence of a network of coordinated water molecules; (3) secondary factors could contribute to stability and come into relevant play in the case of some peptides, such as CB3; (4) it follows that the solutes affecting the structure of water (i.e. the chaotropic effect) could in¯uence peptide and collagen stab-
Stability of Type I Collagen CNBr Peptide Trimers
ility, and also the equilibria among molecular species peptides can form.
Acknowledgments We thank doctors Katharine M. Dyne and Antonella Forlino for suggestions and criticism. Our thanks also to Centro Grandi Strumenti, University of Pavia, for sequencing of peptides a1(I) CB2 and CB2,4 (Dr Alessandra Cobianchi) and for free access to the CD spectropolarimeter. This work was supported by grants from Italian MURST (FAR and 40%) and Consiglio Nazionale delle Ricerche.
References BaÈchinger, H. P. & Davis, J. M. (1991). Sequence speci®c thermal stability of the collagen triple helix. Int. J. Biol. Macromol. 13, 152±156. BaÈchinger, H. P. & Morris, N. P. (1990). Analysis of the thermal stability of type II collagen in various solvents used for reversed-phase high performance chromatography. Matrix, 10, 331± 338. Bella, J., Eaton, M., Brodsky, B. & Berman, H. M. (1994). Crystal and molecular structure of a collagen-like Ê resolution. Science, 266, 75± 81. peptide at 1.9 A Bella, J., Brodsky, B. & Berman, H. M. (1995). Hydration structure of a collagen peptide. Structure, 3, 893 ± 906. Bhatnagar, R. S., Pattabiraman, N., Sorensen, K. R., Langridge, R., MacElroy, R. D. & Renugopalakrishnan, V. (1988). Inter-chain proline:proline contacts contribute to the stability of the triple helical conformation. J. Biomol. Struct. Dynam. 2, 223± 233. Davis, J. M. & BaÈchinger, H. P. (1993). Hysteresis in the triple helix-coil transition of type III collagen. J. Biol. Chem. 268, 25965± 25972. Engel, J., Chen, H., Prockop, D. J. & Klump, H. (1977). 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-ProGly)n and (L-Pro-L-Hyp-Gly)n. Biopolymers, 16, 601 ± 622. Galloway, D. (1982). Appendix: The primary structure. In Collagen in Health and Disease (Weiss, J. B. & Jayson, M. I. V., eds), pp. 528± 557, ChurchillLivingstone, Edinburgh. Jones, E. Y. & Miller, A. (1991). Analysis of structural design features in collagen. J. Mol. Biol. 218, 209 ± 219. Katz, E. P. & David, C. W. (1992). Unique side-chain conformation encoding for chirality and azimuthal orientation in the molecular packing of skin collagen. J. Mol. Biol. 228, 963±969. Long, C. G., Braswell, E., Zhu, D., Apigo, J., Baum, J. & Brodsky, B. (1993). Characterization of collagen-like peptides containing interruptions in the repeating Gly-X-Y sequence. Biochemistry, 32, 11688± 11695. Morris, N. P., Watt, S. L., Davis, J. M. & BaÈchinger, H. P. (1990). Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagen a12a2 and a1a2a3. J. Biol. Chem. 265, 10081± 10087.
Stability of Type I Collagen CNBr Peptide Trimers Phillips, C. L., Morgan, A. L., Lever, L. W. & Wenstrup, R. J. (1992). Sequence analysis of a full-length cDNA for the murine proa2(I) collagen chain: comparison of the derived primary structure with human proa2(I) collagen. Genomics, 13, 1345± 1346. Privalov, P. L. (1982). Stability of proteins. Proteins which do not present a single cooperative system. Advan. Protein Chem. 35, 1± 104. Rossi, A., Vitellaro Zuccarello, L., Zanaboni, G., Monzani, E., Dyne, K. M., Cetta, G. & Tenni, R. (1996). Type I collagen CNBr peptides: species and behavior in solution. Biochemistry, 35, 6048± 6057. Saygin, O., Heidemann, E. & Klump, H. (1978). The triple helix-coil transition of cyanogen-bromide pep-
493 tides of the a1-chain of the calf-skin collagen. Biopolymers, 17, 511± 522. VaÂsquez, M., NeÂmethy, G. & Scheraga, H. A. (1994). Conformational energy calculations on polypeptides and proteins. Chem. Rev. 94, 2183± 2239. Venugopal, M. G., Ramshaw, J. A. M., Braswell, E., Zhu, D. & Brodsky, B. (1994). Electrostatic interactions in collagen-like triple-helical peptides. Biochemistry, 33, 7948± 7956. Vitagliano, L., NeÂmethy, G., Zagari, A. & Scheraga, H. A. (1993). Stabilization of the triple-helical structure of natural collagen by side-chain interactions. Biochemistry, 32, 7354± 7359.
Edited by F. E. Cohen (Received 16 August 1996; received in revised form 24 March 1997; accepted 24 March 1997)
COMMUNICATION
Stability of Type I Collagen CNBr Peptide Trimers Antonio Rossi, Giuseppe Zanaboni, Giuseppe Cetta and Ruggero Tenni* Dipartimento di Biochimica ``Alessandro Castellani'', via Taramelli 3b, University of Pavia, 27100 Pavia, Italy
As indices of triple helix stability of type I collagen CNBr peptide homotrimers, G for monomer-trimer transitions and melting temperatures were obtained from circular dichroism measurements at increasing temperatures. The data were compared with the stability of the parent native molecule. Peptides were found to have a lower stability than the whole collagen molecule. The general implication is that the coordinated water molecules play a key role in determining collagen triple helical stability and high cooperativity at melting. Other factors (monomer stability, ionic and hydrophobic factors, variations of composition, speci®c sequences) could also contribute towards peptide stability; these factors could explain the data obtained in the case of peptide a1(I) CB3. # 1997 Academic Press Limited
*Corresponding author
Keywords: collagen; collagen peptides; triple helix stability; cooperative melting
The stability of the collagen triple helix has been the subject of extensive work. Privalov (1982) has reviewed and discussed this work in terms of the thermodynamics of collagen structure stabilization, its correlation with the chemical characteristics of collagen structure and the participation of water in its stabilization. Hydroxyproline and the extended network of coordinated water molecules are major contributors to collagen stability. Recently, the pivotal role of the Hyp hydroxyl group for the regular water networks has been clearly demonstrated after determination of the molecular and the crystal structures of the peptide (Pro-Hyp-Gly)4-Pro-HypAla-(Pro-Hyp-Gly)5 (Bella et al., 1994, 1995). Other factors involved in collagen structure stabilization have also been reported; these were mainly based on computer modeling: side-chain interactions by Vitagliano et al. (1993); electrostatic interactions (Venugopal et al., 1994; charged tripoles: Katz & David, 1992); hydrophobic interactions (Jones & Miller, 1991; interchain proline-proline contacts: Bhatnagar et al., 1988). For a review on these aspects see VaÂsquez et al. (1994). We recently puri®ed CNBr peptides covering more than 96% of a1(I) and a2(I) chains of acid-soluble type I collagen from calf skin and analysed the molecular species peptides can form, their conAbbreviations used: Hyp, hydroxyproline; CD, circular dichroism; tm, melting temperature. 0022±2836/97/240488±6 $25.00/0/mb971046
formation and thermodynamic and kinetic aspects of equilibria between species. Single-stranded molecules (monomers) of all peptides are able to form homotrimers with triple helical conformation; trimers of some peptides are able to auto-aggregate. The stability of trimers merits futher consideration, because a high fraction of peptide monomers fold into trimers, but full helicity is not reached, and because melting temperatures for peptides are lower than that obtained for native type I collagen molecules (Rossi et al., 1996). Only the largest peptide we analysed, a2(I) CB3,5, showed irreversible denaturation such as occurs for collagen. Using circular dichroism, the positive signal occurring at 221 nm, which is typical of native collagen and trimeric peptides in a triple-helical conformation, was found to increase very slowly with time for this peptide and we found variations after nine months of equilibration at 4 C (data not shown). Since Hyp content and total imino acid content differ for the peptides (ranging from 82 to 167 and 198 to 333 residues per 1000 residues, respectively), we analysed the thermodynamic aspects of peptide trimer stability, to investigate their contribution to the stability of the whole molecule. As reference, we used data cited in Privalov's work and data on type I collagen CNBr peptides (Saygin et al., 1978). We could not make any comparison with type I collagen homotrimer, [a1(I)]3, since thermodynamic data similar to ours have not been reported for this collagen type. # 1997 Academic Press Limited
Stability of Type I Collagen CNBr Peptide Trimers
489
Experimental data were obtained by following the decrease of the CD signal at 221 nm at increasing temperatures (for the procedure used, see Rossi et al., 1996). The denaturation of collagen is a slow process, as is the peptide trimer to monomer conversion (Rossi et al., 1996). The apparent melting curve is therefore more or less close to the equilibrium melting curve. Thus, we used low heating rates, as suggested by Privalov (1982), and comparable with those used in similar experiments by Long et al. (1993) and Venugopal et al. (1994). The (van't Hoff) enthalpy change we obtained for bovine type I collagen melting was 379 kcal molÿ1, quite similar to the value from calorimetric measurements reported for rat skin collagen (Privalov, 1982). Melting temperatures, van't Hoff (or effective) H , S and G were calculated using the procedure of Engel et al. (1977) by using their equations 11, 7, 5 and 4. The structure of the mathematical procedure (the set of formulas) for computing the parametric values is such that the parameters are not independent (H f(c, tm, F); S f(c, tm, H ); G f(H , S ), where c is total monomer concentration and F is the fraction of chains in helical state at each temperature tested). We also obtained data on the monomer drift, i.e. the variation of monomer CD signal with temperature. Stability of peptides Melting temperature and G of the monomertrimer transition are indices of peptide trimer stability. They were found to be correlated (r2 0.993 for all samples analysed; r2 0.994 for peptides from a1(I), with the exclusion of CB3) (data not shown). When normalized per unit length (per triplet), all thermodynamic parameters correlated with Hyp content and, better still, with total imino acid content (Figures 1 and 2). It is evident from Figure 1 that enthalpic factors are the driving forces for the spontaneity of trimer formation, with a concomitant decrease of entropy. The peptide located in the middle of a1(I), CB3, behaves differently in that it shows high ``extra-stability''. This result is not artifactual as it was con®rmed in ®ve different determinations using three different preparations. Standard deviation was <10% of the mean for all thermodynamic parameters. Privalov (1982) assumed that the contribution made by Hyp towards collagen stability is due to two factors: a contribution identical to that of Pro, and a speci®c contribution for Hyp ability to act as the coordination centre of water molecules. The data concerning these contributions have been calculated for peptides and compared with Privalov's data for collagens (Table 1). The ordered water shell contributes towards enthalpic helical stability and greatly decreases entropy for both collagens and peptides. This special Hyp contribution is lower for peptides than for the whole molecule.
Figure 1. Plots for peptide trimer formation (monomertrimer transition) of the thermodynamic parameters, normalized per unit length (per triplet), versus % imino acid content and versus % Hyp content. The following peptides were analysed: a1(I) CB2 - CB8 - CB3 - CB7 CB6 and composite peptides CB2,4 - CB3,7 and CB3,8 (®lled circles, except CB3 (diamond)); a2(I) CB4 - CB3,5 (®lled squares). Open circle, acid-soluble type I collagen. Interpolating lines were for peptides from a1(I) only (continuous lines) or also for peptides from a2(I) (broken lines); r2 is indicated for each interpolation. The data for CB3 and collagen were excluded from all interpolations.
This ®nding can be explained by the presence of a less extended and/or ordered hydration shell. The crossover of all interpolating lines in Figures 1 and 2 with the value 0 of all parameters is near 20% total imino acid content and 10% Hyp content. Thus, a minimum of about 100 Hyp residues per 1000 residues and about 200 residues of Pro Hyp are required for trimer formation for the peptides we analysed. Peptides have a Hyp/ Pro ratio in the range 0.71 to 1.38 (0.81 to 1.18 for peptides from a1(I) only, those from a2(I) being at the extremes of the range). By assuming a ratio of 1:1, the spontaneity of trimer formation at 25 C, i.e. when G just turns negative, can be calculated
490
Stability of Type I Collagen CNBr Peptide Trimers
Figure 2. Plots of melting temperatures (calculated at the formal concentration of 1 mM) of peptide trimers and of the linear variation with temperature of CD signal at 221 nm for peptide monomers. Symbols are as in Figure 1.
from the equations given in Table 1. A minimum content of 200 residues of Hyp Pro per 1000 residues is required both for collagens and peptides. The most important ®nding is the similarity of the general Hyp contribution to G in the case of both peptides and collagens, in concomitance with
a lower water contribution to peptide stability, about 1/3.4 with respect to the value for collagen (see equations for G in Table 1). If we assume that, on average, peptide homotrimers share with their parent native molecule the same characteristic one interchain hydrogen bond and the same average Hyp content and total imino acid content, we can conclude with the more general view that the greatest contribution to the stability of the collagen triple helix is given by the extended and highly ordered hydration shell. This could also explain the very high degree of cooperativity of collagen melting. This last point raises the question of the number and size of cooperative blocks. Type I collagen melts with a very sharp transition and a very large enthalpy change, as measured by calorimetric studies. Calorimetric enthalphy is about 12 times greater than van't Hoff (or effective) enthalphy. Collagen melting was thus interpreted as an extremely cooperative process and the length of each cooperative block, calculated from the ratio calorimetric/van't Hoff enthalpy, is about 1000/12, i.e. about 80 residues per chain or about 250 residues per trimer (Privalov, 1982). Type II collagen has very similar characteristics with a cooperative block length of about 60 residues (BaÈchinger & Morris, 1990). Slightly different values for types I and II collagens are reported by Davis & BaÈchinger (1993), together with data for type III collagen. Other collagens of the ®brillar family show complex transitions (Morris et al., 1990). In this context, we compared our enthalpy data obtained by optical measurements (van't Hoff enthalpies) with calorimetric enthalpy data (Saygin et al., 1978). Unfortunately, the latter data concern only a very few of the peptides we considered (CB2, CB3, CB7 and CB8). We were also unable to ®nd calorimetric data for peptide CB6 that covers a
Table 1. Thermodynamic data of monomer-trimer transition for peptides versus data for collagen H (kcal molÿ1) ÿ 574 ÿ 8.56 ZHyp 930 ÿ 9.87 ZHyp ÿ 551 ÿ 0.74 ZP Hyp ÿ 7.35 ZHyp(w) 1077 ÿ 3.90 ZP Hyp ÿ 3.28 ZHyp(w) S (cal Kÿ1 molÿ1) ÿ 2630 1.43 ZP Hyp ÿ 23.4 ZHyp(w) 3264 ÿ 12.0 ZP Hyp ÿ 9.65 ZHyp(w) G (kcal molÿ1) 215 ÿ 0.43 ZP Hyp ÿ 1.34 ZHyp(w) 103 ÿ 0.32 ZP Hyp ÿ 0.39 ZHyp(w)
r2
Collagen (equation 4 in *) Peptides Collagen (from table V in *) Peptides
0.923 0.946 0.991
Collagen (equation 6 in *) Peptides
0.990
Collagen (equation 7 in *, modi®ed) Peptides (a)
0.996
Data for collagen are from Privalov (1982) (indicated in the Table with a *); data for peptides are from a1(I) only, with the exclusion of CB3. The values are computed for 1000 residues. When based on experimental data, the values which best ®t the equations >parameter a b ZP Hyp c ZHyp(w) are given; in this case, r2 for the linear correlation between experimental and calculated data is given. Z, residues/1000 residues of the imino acid(s) in superscript; Hyp(w), denotes the contribution of Hyp as the coordination centre of water molecules. (a) Due to the signi®cant correlation between G and tm, one could also obtain an estimation of tm from peptide imino acid content by the equation: tm,1000 ( C) ÿ 201 0.52 ZP Hyp 0.98 ZHyp(w) (r2 0.985) and then by: tm ( C) tm,1000 number of residues in the peptide trimer / 1000.
491
Stability of Type I Collagen CNBr Peptide Trimers
stable region at the C-terminal end of the a1(I) chain and for peptides from the a2(I) chain. Three results arise from this comparison: the length of the cooperative blocks we can calculate for peptides is of the same amplitude of those for collagen, the number of cooperative blocks is much lower for peptides, and peptides differ from each other: 1.3 cooperative blocks with 30 residues/block for peptide CB2, 2.0 cooperative blocks with 75 residues/block for CB3, 2.8 blocks with 95 residues/ block for CB7, 2.3 blocks with 120 residues/block for CB8. From these data we have the indication of a higher than average stability for the N-terminal peptide and for a central one (CB2 and CB3, respectively; CB6 probably shares this characteristic), and a lower stability for the other peptides. Furthermore, block length is not dependent on peptide length. For all peptides we can reasonably assume that the very same factor, i.e. the contribution of multiple weak bonds of Hyp/water shell, is a key factor not only towards maintaining stability but also for the modulation of the degree of cooperativity at melting. Such a factor is lower for peptides and therefore other stabilizing factors could become more evident for the stability of certain peptides, like CB3. Stability of CB3 Results of calorimetric measurements showed that CB3 did not present any extra-stability (see Table III and Figure 5 of Saygin et al., 1978). The
extra-stability we saw in our data is thus due to the cooperative block length. The result is therefore the unexpected ®nding of the presence of high triple helical stability or a lower than average block length not only at the terminal ends (BaÈchinger & Davis, 1991), due to their high Hyp and total imino acid content, but also in the central portion spanned by CB3 (residues 403 to 551 of the triple helix). This CB3 stability is not the result of its imino acid content (see Figure 1); so other factors involved have no longer a quantitatively marginal role. A ®rst clue to support the stability of CB3 comes from the calculated relative stability along type I collagen and a1(I) chain (see Figure 2 of BaÈchinger & Davis, 1991): the CB3 region has the highest mean stability. It has also the lowest dispersion around the mean and for this peptide, at variance from all others, no profound minima, i.e. no zones of very low relative stability, are present. Furthermore, the drift of monomer CD signal at increasing temperatures is close to zero for CB3 (Figure 2) and is compatible with its imino acid content. The monomer drift can be read as the variation of the secondary structure that monomers maintain for the presence of rigid imino acids. CB3 monomer is present in the most stable or disordered structure even at 25 C and, therefore, G is the difference between two states (monomer and trimer), the former being already at its energetic minimum. Charged tripoles arise from Gly-Xÿ-Y sequences. Positive and negative tripoles have an or-
Table 2. Amino acid composition of some selected collagen polypeptide chains (residues/1000 residues)
D E H K R Hyp S T Y C G N Q A F I L M P V W
Type I collagen
Whole
26.6 47.7 4.9 32.9 51.3 109.1 31.9 17.1
29.6 48.3 2.0 35.5 50.3 116.4 32.5 16.8
335.3 16.8 24.0 112.1
335.3 12.8 24.7 118.3
12.2 11.2 24.0 6.2 113.4 23.3
11.8 7.9 18.7 6.9 116.4 15.8
a1(I) Gap
Ov.
Whole
a1(I) CB3 Gap
Ov.
Whole
26.8 67.1
18.5 92.6
31.6 52.6
39.7 49.6 101.2 27.8 19.8
23.5 43.1 3.9 31.4 51.0 131.4 37.2 13.7
33.6 40.3 114.1 20.1
55.6 37.0 92.6
21.1 42.1 126.3 31.6
36.5 41.7 5.2 26.0 57.3 114.6 52.1 20.4
335.3 11.9 19.8 140.9 (49%) 6.0 6.0 9.9 6.0 119.0 17.9
335.3 13.7 29.4 96.1 (53%) 17.6 9.8 27.5 7.8 113.7 13.7
335.6 13.4 40.3 147.7
333.3 37.0 166.7 (33%) 18.5
336.8 21.1 42.1 136.8 (62%) 21.1
111.1 37.0
31.6 10.5 73.2 21.1
35.7 53.6
20.1 20.1 6.7 87.2 26.8
333.3 5.2 20.8 93.8 5.2 15.6 20.8 140.6 10.4
a1(I) CB6 Gap 39.2 68.6 29.4 58.8 88.2 39.2 29.4 333.3 19.6 127.5 (54%) 9.8 9.8 127.5 19.6
Ov. 33.3 11.1 11.1 22.2 55.6 144.4 66.7 11.1 333.3 11.1 22.2 55.6 (20%) 11.1 22.2 33.3 155.6
The composition was calculated from bovine a1(I) and a2(I) sequences, where known; otherwise, human sequences were used (Galloway, 1982; Phillips et al., 1992). Characters in italics indicate difference with a1(I); in bold indicate a difference between gap and overlap zone. The values in parenthesis are the percentage of alanine in X position. Positioning of residues in gap and overlap (Ov.) zones. CB3: gap, residues 403 to 456 (54 res. long); overlap, res. 457 to 551 (95 res.). CB6: gap, res. 823 to 924 (102 res.); overlap, res. 925 to 1014 (90 res.) (from Jones & Miller, 1991)
492 dered relative disposition along the triple helix and this contributes to triple helix chirality and to the azimuthal orientation of adjacent triple helices (Katz & David, 1992). They can also contribute to collagen and CB3 stability. Four out of 14 negative tripoles, and probably the same fraction of positive ones, fall into the CB3 region (Katz & David, 1992). Other, but unquanti®able, clues come from the composition of the peptides as a whole or of their portions that fall into gap and overlap zones of the ®brils (Table 2). This difference among the two zones is here brought in evidence because, by applying the equation in Table 1 that relates G with Hyp and total imino acid contents, it can be calculated that by far the greatest contribution to stability is given by the portions falling into the overlap zones. Several amino acids are differently represented in the two zones of a1(I) chain and of CB3 and CB6. We performed this calculation only for these two peptides because in the simplest way they start in the gap zone and end in the overlap one; other peptides have a more complex disposition in the two zones. Some amino acids, apart from Pro and Hyp, have a preferential X or Y position, but there are not signi®cant differences between the two zones for any amino acid, with the relevant exception of alanine. When isolated chains fold into trimers, hydrophobic amino acids have a decreased area in contact with the solvent, especially when they are in the Y position (Jones & Miller, 1991). Thus, it may be relevant that the CB3 gap zone is devoid of the largest amino acids of this class. Rather, the CB3 gap zone is particularly more rich in proline residues than the overlap zone is, differing from a1(I) and CB6, and in glutamic acid and lysine. However, a much more complex picture emerges if one takes into account the composition of the stretch in a2(I) corresponding to CB3 in a1(I). But this stretch is also poor in Hyp and total imino acid content (data not shown), and therefore the stability of the central region of type I collagen is not brought about exclusively by the presence of a2(I) chain.
Conclusive remarks Type I collagen CNBr peptides are able to form (homo)trimers. Here we have presented thermodynamic data concerning the stability of peptide trimers in comparison with the stability of the whole molecule. We conclude that: (1) peptides have a lower stability than the parent molecule, mainly because of a less extended and/or structured water shell; (2) this implies that the key feature involved in collagen triple helical stability and in the very high cooperativity at melting is the presence of a network of coordinated water molecules; (3) secondary factors could contribute to stability and come into relevant play in the case of some peptides, such as CB3; (4) it follows that the solutes affecting the structure of water (i.e. the chaotropic effect) could in¯uence peptide and collagen stab-
Stability of Type I Collagen CNBr Peptide Trimers
ility, and also the equilibria among molecular species peptides can form.
Acknowledgments We thank doctors Katharine M. Dyne and Antonella Forlino for suggestions and criticism. Our thanks also to Centro Grandi Strumenti, University of Pavia, for sequencing of peptides a1(I) CB2 and CB2,4 (Dr Alessandra Cobianchi) and for free access to the CD spectropolarimeter. This work was supported by grants from Italian MURST (FAR and 40%) and Consiglio Nazionale delle Ricerche.
References BaÈchinger, H. P. & Davis, J. M. (1991). Sequence speci®c thermal stability of the collagen triple helix. Int. J. Biol. Macromol. 13, 152±156. BaÈchinger, H. P. & Morris, N. P. (1990). Analysis of the thermal stability of type II collagen in various solvents used for reversed-phase high performance chromatography. Matrix, 10, 331± 338. Bella, J., Eaton, M., Brodsky, B. & Berman, H. M. (1994). Crystal and molecular structure of a collagen-like Ê resolution. Science, 266, 75± 81. peptide at 1.9 A Bella, J., Brodsky, B. & Berman, H. M. (1995). Hydration structure of a collagen peptide. Structure, 3, 893 ± 906. Bhatnagar, R. S., Pattabiraman, N., Sorensen, K. R., Langridge, R., MacElroy, R. D. & Renugopalakrishnan, V. (1988). Inter-chain proline:proline contacts contribute to the stability of the triple helical conformation. J. Biomol. Struct. Dynam. 2, 223± 233. Davis, J. M. & BaÈchinger, H. P. (1993). Hysteresis in the triple helix-coil transition of type III collagen. J. Biol. Chem. 268, 25965± 25972. Engel, J., Chen, H., Prockop, D. J. & Klump, H. (1977). 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-ProGly)n and (L-Pro-L-Hyp-Gly)n. Biopolymers, 16, 601 ± 622. Galloway, D. (1982). Appendix: The primary structure. In Collagen in Health and Disease (Weiss, J. B. & Jayson, M. I. V., eds), pp. 528± 557, ChurchillLivingstone, Edinburgh. Jones, E. Y. & Miller, A. (1991). Analysis of structural design features in collagen. J. Mol. Biol. 218, 209 ± 219. Katz, E. P. & David, C. W. (1992). Unique side-chain conformation encoding for chirality and azimuthal orientation in the molecular packing of skin collagen. J. Mol. Biol. 228, 963±969. Long, C. G., Braswell, E., Zhu, D., Apigo, J., Baum, J. & Brodsky, B. (1993). Characterization of collagen-like peptides containing interruptions in the repeating Gly-X-Y sequence. Biochemistry, 32, 11688± 11695. Morris, N. P., Watt, S. L., Davis, J. M. & BaÈchinger, H. P. (1990). Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagen a12a2 and a1a2a3. J. Biol. Chem. 265, 10081± 10087.
Stability of Type I Collagen CNBr Peptide Trimers Phillips, C. L., Morgan, A. L., Lever, L. W. & Wenstrup, R. J. (1992). Sequence analysis of a full-length cDNA for the murine proa2(I) collagen chain: comparison of the derived primary structure with human proa2(I) collagen. Genomics, 13, 1345± 1346. Privalov, P. L. (1982). Stability of proteins. Proteins which do not present a single cooperative system. Advan. Protein Chem. 35, 1± 104. Rossi, A., Vitellaro Zuccarello, L., Zanaboni, G., Monzani, E., Dyne, K. M., Cetta, G. & Tenni, R. (1996). Type I collagen CNBr peptides: species and behavior in solution. Biochemistry, 35, 6048± 6057. Saygin, O., Heidemann, E. & Klump, H. (1978). The triple helix-coil transition of cyanogen-bromide pep-
493 tides of the a1-chain of the calf-skin collagen. Biopolymers, 17, 511± 522. VaÂsquez, M., NeÂmethy, G. & Scheraga, H. A. (1994). Conformational energy calculations on polypeptides and proteins. Chem. Rev. 94, 2183± 2239. Venugopal, M. G., Ramshaw, J. A. M., Braswell, E., Zhu, D. & Brodsky, B. (1994). Electrostatic interactions in collagen-like triple-helical peptides. Biochemistry, 33, 7948± 7956. Vitagliano, L., NeÂmethy, G., Zagari, A. & Scheraga, H. A. (1993). Stabilization of the triple-helical structure of natural collagen by side-chain interactions. Biochemistry, 32, 7354± 7359.
Edited by F. E. Cohen (Received 16 August 1996; received in revised form 24 March 1997; accepted 24 March 1997)