Sequence Dependence of the Folding of Collagen-like Peptides
1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês
10.1074/jbc.274.12.7668
ISSN1083-351X
AutoresMichael S. Ackerman, Manjiri Bhate, Nigel Shenoy, Konrad Beck, John A. M. Ramshaw, Barbara Brodsky,
Tópico(s)Chemical Synthesis and Analysis
ResumoThe refolding of thermally denatured model collagen-like peptides was studied for a set of 21 guest triplets embedded in a common host framework: acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide. The results show a strong dependence of the folding rate on the identity of the guest Gly-Xaa-Yaa triplet, with the half-times for refolding varying from 6 to 110 min (concentration = 1 mg/ml). All triplets of the form Gly-Xaa-Hyp promoted rapid folding, with the rate only marginally dependent on the residue in the Xaa position. In contrast, triplets of the form Gly-Pro-Yaa and Gly-Xaa-Yaa were slower and showed a wide range of half-times, varying with the identity of the residues in the triplet. At low concentrations, the folding can be described by third-order kinetics, suggesting nucleation is rate-limiting. Data on the relative nucleation ability of different Gly-Xaa-Yaa triplets support the favorable nature of imino acids, the importance of hydroxyproline, the varying effects of the same residue in the Xaa position versus the Yaa position, and the difficulties encountered when leucine or aspartic acid are in the Yaa position. Information on the relative propensities of different tripeptide sequences to promote nucleation of the triple-helix in peptides will aid in identification of nucleation sites in collagen sequences. The refolding of thermally denatured model collagen-like peptides was studied for a set of 21 guest triplets embedded in a common host framework: acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide. The results show a strong dependence of the folding rate on the identity of the guest Gly-Xaa-Yaa triplet, with the half-times for refolding varying from 6 to 110 min (concentration = 1 mg/ml). All triplets of the form Gly-Xaa-Hyp promoted rapid folding, with the rate only marginally dependent on the residue in the Xaa position. In contrast, triplets of the form Gly-Pro-Yaa and Gly-Xaa-Yaa were slower and showed a wide range of half-times, varying with the identity of the residues in the triplet. At low concentrations, the folding can be described by third-order kinetics, suggesting nucleation is rate-limiting. Data on the relative nucleation ability of different Gly-Xaa-Yaa triplets support the favorable nature of imino acids, the importance of hydroxyproline, the varying effects of the same residue in the Xaa position versus the Yaa position, and the difficulties encountered when leucine or aspartic acid are in the Yaa position. Information on the relative propensities of different tripeptide sequences to promote nucleation of the triple-helix in peptides will aid in identification of nucleation sites in collagen sequences. circular dichroism The collagen triple helix is the basic structural motif found in all fibril-forming collagens as well as some host-defense proteins such as C1q, mannose-binding protein, and macrophage scavenger receptor (1Brodsky B. Ramshaw J.A.M. Matrix Biol. 1997; 15: 545-554Crossref PubMed Scopus (405) Google Scholar,2Beck K. Brodsky B. J. Struct. Biol. 1998; 122: 17-29Crossref PubMed Scopus (274) Google Scholar). The triple-helix conformation consists of three extended polyproline II-like chains supercoiled around each other as determined by x-ray fiber diffraction, crystallography, and NMR (3Rich A. Crick F.H.C. J. Mol. Biol. 1961; 3: 483-506Crossref PubMed Scopus (601) Google Scholar, 4Fraser R.D.B. MacRae T.P. Suzuki E. J. Mol. Biol. 1979; 129: 463-481Crossref PubMed Scopus (285) Google Scholar, 5Bella J. Eaton M. Brodsky B. Berman H.M. Science. 1994; 266: 75-81Crossref PubMed Scopus (890) Google Scholar, 6Mayo K.H. Biopolymers. 1996; 40: 359-370Crossref PubMed Scopus (42) Google Scholar, 7Baum J. Brodsky B. Folding & Design. 1997; 2: R53-R60Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The three chains are staggered by one residue with respect to each other and stabilized by interchain hydrogen bonding (5Bella J. Eaton M. Brodsky B. Berman H.M. Science. 1994; 266: 75-81Crossref PubMed Scopus (890) Google Scholar, 8Privalov P. Adv. Protein Chem. 1982; 35: 1-104Crossref PubMed Scopus (944) Google Scholar, 9Bella J. Brodsky B. Berman H.M. Structure. 1995; 3: 893-906Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). This conformation requires that every third residue must be a glycine, generating a repeating (Gly-Xaa-Yaa)n pattern, and that a high proportion of residues are the imino acids proline and hydroxyproline. Gly-Pro-Hyp is indeed the most common and stabilizing tripeptide found in collagens. 1Standard three-letter nomenclature is used to describe peptide sequences with Hyp representing 4-hydroxyproline. In the standard one-letter code for amino acids, O is used to represent 4-hydroxyproline. Host-guest peptides are named by the sequence of their guest triplet. The folding of the collagen triple helix in vivo is a multistep process involving chain association, registration, nucleation, and propagation (10Engel J. Prockop D.J. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 137-152Crossref PubMed Scopus (269) Google Scholar, 11McLaughlin S.H. Bulleid N.J. Matrix Biol. 1997; 16: 369-377Crossref Scopus (98) Google Scholar, 12Bulleid N.J. Cell. Dev. Biol. 1996; 7: 667-672Google Scholar). There is also evidence for the involvement of chaperones (13Wilson R. Lee J.F. Bulleid N.J. J. Biol. Chem. 1998; 273: 9637-9643Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 14Nagata K. Trends. Biochem. Sci. 1996; 21: 23-26Abstract Full Text PDF Scopus (254) Google Scholar). Fibril-forming collagens are synthesized at the rough endoplasmic reticulum membrane in a precursor form, procollagen, containing both N- and C-terminal propeptides terminating the long central triple helix. Proper chain selection and registration is initiated by the association of the C-propeptide domains into trimers followed by nucleation of the correctly aligned triple helix (15Lees J.F. Tasab M. Bulleid N.J. EMBO J. 1997; 16: 908-916Crossref PubMed Scopus (125) Google Scholar, 16Bulleid N.J. Dalley J.A. Lees J.F. EMBO J. 1997; 16: 6694-6701Crossref PubMed Scopus (87) Google Scholar) and propagation in a C- to N-terminal direction (17Bächinger H.P. Bruckner P. Timpl R. Prockop D.J. Engel J. Eur. J. Biochem. 1980; 106: 619-632Crossref PubMed Scopus (204) Google Scholar). In the unfolded state, most proline residues in the Yaa position of Gly-Xaa-Yaa triplets are enzymatically hydroxylated, and the resulting hydroxyproline (Hyp) residues are required for the formation and stabilization of the triple helix (18Sakakibara S. Inouye K. Shudo K. Kishida Y. Kobayashi Y. Prockop D.J. Biochim. Biophys. Acta. 1973; 303: 198-202Crossref PubMed Scopus (271) Google Scholar). Folding studies on mature collagens are complicated by their length and varied sequences; these complicating features can be reduced by the use of natural or synthetic peptides. Collagen fragments have been used to better define the folding process, e.g. the observation of third-order kinetics for a 36-residue cyanogen bromide fragment of collagen type I (19Piez K.A. Sherman M.S. Biochemistry. 1970; 9: 4134-4140Crossref PubMed Scopus (56) Google Scholar). Synthetic peptide models of the triple helix allow the sequence dependence of folding to be investigated systematically by varying both the design and the composition of the Gly-Xaa-Yaa sequences. Investigations on synthetic model peptides, such as (Pro-Pro-Gly)n, which adopt a stable triple-helical structure, have allowed quantitation of third-order rate constants, the effect of length and the testing of sophisticated theoretical models including folding intermediates resulting from incorrectly staggered chains (20Sutoh K. Noda H. Biopolymers. 1974; 13: 2477-2488Crossref PubMed Scopus (20) Google Scholar,21Weidner H. Biopolymers. 1975; 14: 763-780Crossref PubMed Scopus (10) Google Scholar). Here we present data on the refolding of a set of homologous peptides that contain one variable Gly-Xaa-Yaa guest triplet embedded in a Gly-Pro-Hyp-rich host sequence. This design allows the analysis of the effect of a single triplet sequence on different triple-helix properties. Work in our laboratory has shown that all host-guest peptides analyzed so far form stable triple helices, with melting temperatures dependent on the identity of the guest triplet (22Shah N.K. Ramshaw J.A.M. Kirkpatrick A. Shah C. Brodsky B. Biochemistry. 1996; 35: 10262-10268Crossref PubMed Scopus (117) Google Scholar, 23Chan V.C. Ramshaw J.A.M. Kirkpatrick A. Beck K. Brodsky B. J. Biol. Chem. 1997; 272: 31441-31446Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 24Yang W. Chan V.C. Kirkpatrick A. Ramshaw J.A.M. Brodsky B. J. Biol. Chem. 1997; 272: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). This study reports folding rates for a set of host-guest peptides that provide information on the relative propensities of different tripeptide sequences to promote nucleation of the triple helix. Peptides were synthesized on an Applied BioSystems 430A synthesizer using the standard FastMoc (Applied BioSystems) method on a N-(9-fluorenyl)methoxycarbonyl (Fmoc)-RINK resin as described previously (22, 23). Peptides were purified to >90% purity by reversed-phase high performance liquid chromatography on a C-18 column eluted with a binary 10–30% (v/v) acetonitrile/water gradient containing 0.1% trifluoroacetic acid. Peptide identity was confirmed by laser desorption mass spectrometry and amino acid analysis. Peptides were placed in vacuoover P2O5 for more than 48 h before weighing, dissolved in phosphate-buffered saline (10 mmsodium phosphate, 150 mm NaCl, pH 7.0), and stored at 4 °C. Measurements were performed on an AVIV Model 62DS CD2spectrophotometer equipped with a thermoelectric temperature control in 1-mm-path length quartz cells. Peptides were denatured in glass test tubes at 70 °C for 20 min and then rapidly cooled to 15 °C, unless otherwise specified, by quenching in an ice-water bath before transfer into the cuvette, kept at 15 °C. Quenching time was ∼5 s, and the time needed for sample transfer was ∼15 s. The ellipticity at 225 nm was monitored as a function of time over at least 30 min and until the fraction of folded peptides exceeded 0.5 (see below), with data intervals and averaging times of 1 to 60 s, depending on concentration and folding speed. Experiments repeated for some peptides indicated a deviation between their half-times of less than 10%. The fraction of folded peptide (F) is defined as F=(θobs−θm)/(θt−θm)Equation 1 where θobs, θt, and θmrepresent the observed, the triple helix, and monomer ellipticity, respectively. θt was measured directly before denaturation at the temperature used for refolding. θm was determined by extrapolating the initial data points to time zero. This θmvalue was slightly lower than that resulting from linear extrapolation of the monomer ellipticity observed in the high temperature region of equilibrium melting curves, but the use of either value gave similar results. To compare the data of different peptides independent of the folding mechanism, the time (t 12) at which F = 0.5 was determined. The concentration of monomer [A] at any given time was calculated as [A]=(1−F)[A0]Equation 2 with [A]0 denoting the initial monomeric peptide concentration, which was assumed to be equal to the total peptide concentration. Data were fitted to a single-step first (Eq. 3)-, second (Eq. 4)-, or third (Eq. 5)-order kinetics: ln[A]=ln[A0]−k1tEquation 3 1/[A]=1/[A0]+2k2tEquation 4 1/[A]2=1/[A0]2+6k3tEquation 5 Rate constants k i were calculated after linearization from the slope resulting from linear least squares fit. Curves were categorized as of ith order based on maximum linear correlation coefficients. Host-guest peptides of the form acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide provide a useful template to evaluate the contribution of individual Gly-Xaa-Yaa triplets to triple-helix properties (22Shah N.K. Ramshaw J.A.M. Kirkpatrick A. Shah C. Brodsky B. Biochemistry. 1996; 35: 10262-10268Crossref PubMed Scopus (117) Google Scholar, 23Chan V.C. Ramshaw J.A.M. Kirkpatrick A. Beck K. Brodsky B. J. Biol. Chem. 1997; 272: 31441-31446Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 24Yang W. Chan V.C. Kirkpatrick A. Ramshaw J.A.M. Brodsky B. J. Biol. Chem. 1997; 272: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). To assure formation of a stable triple-helix, the guest triplet is flanked by stabilizing Gly-Pro-Hyp triplets. The N and C termini are blocked by acetylation and amidation, respectively, to ensure that the only ionizable groups, if any, would be those introduced in the guest triplet and to eliminate charge repulsion at the ends of the triple helix. The peptide length is designed to be short enough so that the effects of a single guest triplet would not be masked by the constant part of the structure but long enough to ensure triple-helix stability (22Shah N.K. Ramshaw J.A.M. Kirkpatrick A. Shah C. Brodsky B. Biochemistry. 1996; 35: 10262-10268Crossref PubMed Scopus (117) Google Scholar). Because imino acids are found at high frequency in triple helices, guest triplets of the form Gly-Xaa-Hyp, Gly-Pro-Yaa, and Gly-Xaa-Yaa were considered, with the Xaa and Yaa residues occupied by the most common nonpolar residues, and charged residues found in collagens. In the following, we refer to the different peptides by their guest triplet sequence. This design allows analysis of the effects on folding of a single residue within a defined triple-helical environment. CD measurements of all host-guest peptides indicate triple-helical structures at low temperature. The spectra show a characteristic maximum near 225 nm with a mean residue ellipticity in the order of 4,000 deg cm2 dmol−1, which decreases upon unfolding (Fig. 1, inset). Equilibrium unfolding curves exhibit a highly cooperative behavior with melting temperatures ranging from 20 to 45 °C, depending on the identity of the guest triplet (22Shah N.K. Ramshaw J.A.M. Kirkpatrick A. Shah C. Brodsky B. Biochemistry. 1996; 35: 10262-10268Crossref PubMed Scopus (117) Google Scholar, 23Chan V.C. Ramshaw J.A.M. Kirkpatrick A. Beck K. Brodsky B. J. Biol. Chem. 1997; 272: 31441-31446Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 24Yang W. Chan V.C. Kirkpatrick A. Ramshaw J.A.M. Brodsky B. J. Biol. Chem. 1997; 272: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 25Ramshaw J.A.M. Shah N.S. Brodsky B. J. Struct. Biol. 1998; 122: 86-91Crossref PubMed Scopus (295) Google Scholar). The curves can be fitted to a two-state trimer to monomer transition, an assumption supported by analytical ultracentrifugation experiments performed on closely related peptides (26Long C.G. Braswell E. Zhu D. Apigo J. Baum J. Brodsky B. Biochemistry. 1993; 32: 11688-11695Crossref PubMed Scopus (144) Google Scholar). Refolding rates were measured for a total of 21 host-guest peptides (concentration = 1 mg/ml). Despite the variations in stability of the host-guest peptides, a common folding temperature of 15 °C was selected. At this temperature all peptides showed a fraction of folded peptide close to one in their equilibrium melting curves, and little dependence of the folding rate on temperature of folding was observed at 5, 10, 15, and 20 °C (data not shown). As an example, the signal recovery for peptide Gly-Ala-Hyp at 15 °C is shown in Fig. 1, which also illustrates the determination of t 12values as a common measure to compare the refolding behavior of the different peptides. Thet 12 values for the host-guest peptides varied between 6 and 110 min, revealing that the folding rate critically depends on the sequence of the guest triplet (TableI). For example, the folding half-times of peptides Gly-Pro-Hyp, Gly-Ala-Hyp, Gly-Pro-Ala, and Gly-Ala-Ala are 6.0, 8.3, 12, and 21 min, respectively, at 15 °C (Fig.2). The fast folding of Gly-Pro-Hyp is decreased slightly by the substitution of Pro by Ala in the Xaa position and somewhat more when the Hyp is replaced by an Ala.Table ISequence dependence of refoldingGly-Xaa-Hypt 1/2Gly-Pro-Yaat 1/2Gly-Xaa-Yaat 1/2Gly-Pro-Hyp6.0Gly-Pro-Pro11Gly-Ala-Hyp8.3Gly-Pro-Ala12Gly-Ala-Ala21Gly-Leu-Hyp12Gly-Pro-Leu40Gly-Leu-Ala35Gly-Ala-Leu86Gly-Asn-Hyp8.3Gly-Pro-Asn38Gly-Asp-Hyp8.6Gly-Pro-Asp98Gly-Asp-Ala83Gly-Gln-Hyp9.4Gly-Pro-Gln22Gly-Glu-Hyp6.1Gly-Pro-Glu27Gly-Pro-Lys29Gly-Pro-Arg15Gly-Ala-Arg15Refolding of the indicated peptides (concentration = 1 mg/ml) after denaturation at 70 °C for 20 min was monitored at 15 °C, and half-times t 1/2 (in min) were determined. Peptides are sorted in complementary sets. Open table in a new tab Refolding of the indicated peptides (concentration = 1 mg/ml) after denaturation at 70 °C for 20 min was monitored at 15 °C, and half-times t 1/2 (in min) were determined. Peptides are sorted in complementary sets. Guest triplets with Hyp in the Yaa position (Gly-Xaa-Hyp) fold fastest and show only a small dependence on the identity of the residue in the Xaa position. Gly-Pro-Yaa guest triplets with Pro in the Xaa position show a larger range of folding rates and a stronger influence of the identity of the Yaa residue. In the Gly-Pro-Yaa peptides, the residues glutamine and glutamic acid have a very similar influence on the kinetic behavior (t 12 = 22 min for Gln and 27 min for Glu), whereas an exchange of aspartic acid for asparagine drastically slows down the refolding (t 12 = 38 min for Asn and 98 min for Asp). The combination of nonimino acids in both the Xaa and Yaa positions (Gly-Xaa-Yaa) leads to a broad distribution oft 12 values, with peptides Gly-Ala-Leu and Gly-Asp-Ala folding particularly slowly. In general, leucine was observed to decrease the folding rate compared with Ala, with the effect being most striking in the Yaa position. To understand the influence of the single guest triplet on the folding mechanism, the folding data at 1 mg/ml was analyzed by fitting to first-, second-, and third-order kinetics (Equations Equation 3, Equation 4, Equation 5). Considering the full folding process, some peptides showed best fits when assuming a first-order kinetics, whereas others showed best fits when either second- or third-order kinetics were assumed. Analyses of the folding curves of several peptides in which only the late data points were fitted to third-order kinetics (Equation 5) revealed that these regions show a reasonably straight line in 1/[A]02(t) plots (data not shown). This suggests that at a sufficiently low monomer concentration, the third-order step, which relates to chain association and/or nucleation, becomes the rate-limiting step for trimer formation. Folding via a third-order kinetic process should show a profound dependence on concentration, whereas the first-order kinetics of propagation should be independent of concentration. Measurements of the kinetics of folding for a set of 8 peptides at a concentration of 0.4 mg/ml (Fig. 3 A) indicated that at this lower concentration all but the very early time points (t < 10 to 300 s) followed a straight line when plotted as 1/[A]02 (t), giving a good fit to third-order kinetics (Fig. 3 B). This indicates that at a sufficiently low monomer concentration, chain association/nucleation becomes the rate-limiting kinetic step in triple-helix folding. The apparent third-order rate constants derived from the slopes varied from 470 to 16,000 m−2 s−1, with S.D. of approximately ± 10%. The lowest third-order rate constants were found for peptides Gly-Ala-Leu and Gly-Pro-Asp, and the largest was found for Gly-Pro-Hyp (Table II). The Gly-Pro-Pro peptide had a slower folding rate than Gly-Pro-Hyp, showing the effect of a hydroxyl group at one position. The large variation in the rate constants suggests that for these relatively short peptides, the chain association/nucleation step critically depends on the identity of the single guest triplet, and at this low concentration, any other processes like helix propagation have only a marginal contribution.Table IIEffect of sequence on third-order rate constantsPeptidek 3m−2 s−1Gly-Pro-Hyp16,000Gly-Pro-Pro12,000Gly-Ala-Hyp6,000Gly-Pro-Ala4,700Gly-Leu-Ala1,500Gly-Ala-Ala950Gly-Ala-Leu500Gly-Pro-Asp470Refolding of the indicated peptides (concentration = 0.4 mg/ml) after denaturation at 70 °C for 20 min was monitored at 15 °C. Third-order rate constants k 3 were determined according to Equation 5 as outlined in Fig. 4 B. Open table in a new tab Refolding of the indicated peptides (concentration = 0.4 mg/ml) after denaturation at 70 °C for 20 min was monitored at 15 °C. Third-order rate constants k 3 were determined according to Equation 5 as outlined in Fig. 4 B. The folding of collagen molecules in vivo is an intricate and coordinated process (10Engel J. Prockop D.J. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 137-152Crossref PubMed Scopus (269) Google Scholar, 12Bulleid N.J. Cell. Dev. Biol. 1996; 7: 667-672Google Scholar, 27Brodsky B. Gierasch L. King J. The Protein Folding Problem. American Association for the Advancement of Science, Washington, D. C.1990: 55-62Google Scholar). The formation of a trimer requires association and registration of three chains, which is mediated for fibril-forming collagens by the globular C-terminal propeptide domain (10Engel J. Prockop D.J. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 137-152Crossref PubMed Scopus (269) Google Scholar, 11McLaughlin S.H. Bulleid N.J. Matrix Biol. 1997; 16: 369-377Crossref Scopus (98) Google Scholar, 12Bulleid N.J. Cell. Dev. Biol. 1996; 7: 667-672Google Scholar) (Fig. 4). C-propeptide trimerization constrains the three chains at the C terminus such that nucleation can occur at the C terminus of the triple-helical region. Nucleation is considered to be the process in which a series of tripeptide units from the three chains adopt appropriate collagen-like ϕ,ψ angles and form interchain hydrogen bonds. At the C termini of fibril-forming collagens are 5–6 sequential triplets of the form Gly-Xaa-Hyp (often Gly-Pro-Hyp units), which are thought to constitute all or part of the nucleation site. For example, in collagen type III, C-terminal Gly-Xaa-Hyp triplets are required for nucleation, and deletion experiments showed that as long as two Gly-X-Hyp triplets are retained, nucleation is effective, and triple-helix folding is complete (16Bulleid N.J. Dalley J.A. Lees J.F. EMBO J. 1997; 16: 6694-6701Crossref PubMed Scopus (87) Google Scholar). Following nucleation, propagation proceeds in a zipper-like manner from the C to N terminus. The rate-limiting step of propagation is cis-transisomerization at imino acid peptide bonds, which have a significant proportion of cis bonds in unfolded chains (17Bächinger H.P. Bruckner P. Timpl R. Prockop D.J. Engel J. Eur. J. Biochem. 1980; 106: 619-632Crossref PubMed Scopus (204) Google Scholar, 28Bächinger H.P. Bruckner P. Timpl R. Engel J. Eur. J. Biochem. 1978; 90: 605-613Crossref PubMed Scopus (101) Google Scholar, 29Bächinger H.P. J. Biol. Chem. 1987; 262: 17144-17148Abstract Full Text PDF PubMed Google Scholar). The studies reported here on a set of host-guest peptides demonstrate that the folding rate of the triple helix critically depends on the sequence of a single guest triplet. Analysis of our data indicates that the folding proceeds via a mechanism involving more than a single reaction step and that the folding involves a third-order process that becomes rate-limiting at low concentrations. The steps requiring the involvement of three polypeptide chains are chain association and nucleation. Although the association process, which is limited by diffusion, is unlikely to be significantly affected by sequence, triple-helix nucleation is known to be facilitated by the presence of conformationally restricted imino acids and is thus expected to be sequence-dependent (30Von Hippel P.H. Harrington W.F. Biochim. Biophys. Acta. 1959; 36: 427-447Crossref PubMed Scopus (38) Google Scholar) (Fig. 4). In the present study, the magnitude of the third-order rate constant is strongly affected by the identity of the guest triplet and is greater for imino acid-containing triplets, suggesting it is the nucleation step itself reflected by these values. NMR studies on peptides with specific 15N labels indicated that nucleation can occur at (Gly-Pro-Hyp)nsites at either end of a peptide (31Liu X. Siegel D.L. Fan P. Brodsky B. Baum J. Biochemistry. 1996; 35: 4306-4313Crossref PubMed Scopus (48) Google Scholar). For the Gly-Pro-Hyp-enriched host-guest peptides, it is realistic to assume that nucleation could begin at any tripeptide unit in the chain (32Weidner H. Engel J. Fietzek P.P. Blout E.R. Bovey F.A. Goodman M. Lotan N. Peptides, Polypeptides, and Proteins. Wiley, New York1974: 419-435Google Scholar) (Fig. 4). Previous findings suggest the nucleation domain is as long as six triplets in noncovalently linked peptides (20Sutoh K. Noda H. Biopolymers. 1974; 13: 2477-2488Crossref PubMed Scopus (20) Google Scholar, 21Weidner H. Biopolymers. 1975; 14: 763-780Crossref PubMed Scopus (10) Google Scholar), making this a dominant event in short peptides. The third-order rate constants and half-times of folding yield information concerning the relative propensity of different residues in the Xaa and Yaa position to initiate triple-helix nucleation. Entropic factors are likely to play an important role in nucleation because imino acids are sterically constrained to dihedral angles similar to those found in collagen. Gly-Pro-Hyp is the fastest folding triplet, and all Gly-Xaa-Hyp triplets are very favorable. The Hyp residue appears more favorable than Pro, as seen in the faster folding rate of Gly-Pro-Hyp versus Gly-Pro-Pro. It has been suggested that the OH of Hyp has an inductive effect, leading to a decrease in thecis:trans isomer ratio compared with Pro in the unfolded state (33Eberhardt E.S. Panasik Jr., N. Raines R.T. J. Am. Chem. Soc. 1996; 118: 12261-12266Crossref PubMed Scopus (199) Google Scholar). A decrease in the cis isomer concentration could accelerate the propagation step. In addition, the decreased cis:trans ratio could promote nucleation by making it more likely to find a stretch of contiguous all-trans tripeptide units and by creating a more rigid monomer chain (34Bansal M. Ananthanarayanan V.S. Biopolymers. 1988; 27: 299-312Crossref PubMed Scopus (16) Google Scholar). Although Pro in the Xaa position also can lead to favorable folding, the identity of the nonimino acid residue in the Yaa position of Gly-Pro-Yaa triplets has a very strong influence. For example, Gly-Pro-Ala is a fast folding peptide, whereas Gly-Pro-Asp has the slowest folding rate observed. In addition to entropic factors, the influence of specific side chains in promoting nucleation may relate to steric factors, electrostatic interactions, and hydrogen bonding. The difficulty in packing bulky residues such as Leu in the Yaa position (35Bansal M. Int. J. Pept. Protein Res. 1977; 9: 224-234Crossref PubMed Scopus (23) Google Scholar), which is less exposed than the Xaa position, may contribute to the slow folding of Gly-Pro-Leu and Gly-Ala-Leu peptides. Despite its large side chain, arginine in the Yaa position is favorable in promoting chain nucleation as well as for stabilization (24Yang W. Chan V.C. Kirkpatrick A. Ramshaw J.A.M. Brodsky B. J. Biol. Chem. 1997; 272: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and both features may be related to its ability to form multiple hydrogen bonds combined with its restricted mobility (36Richardson J.S. Richardson D. Fasman G.D. Prediction of Protein Structure and the Principles of Protein Conformation. Plenum Publishing Corp., New York1989: 1-98Crossref Google Scholar, 37Vitagliano L. Nemethy G. Zagari A. Scheraga H.A. Biochemistry. 1993; 32: 7354-7359Crossref PubMed Scopus (54) Google Scholar). Gly-Pro-Asp is the slowest folding peptide, suggesting an unfavorable effect of aspartic acid in the Y position. It was previously observed that aspartic acid in the Yaa position had a destabilizing effect on the triple helix. Both the decreased folding rate and low stability may be related to the restricted rotational freedom of Asp in the triple helix, hindering its participation in interchain hydrogen bond formation (23Chan V.C. Ramshaw J.A.M. Kirkpatrick A. Beck K. Brodsky B. J. Biol. Chem. 1997; 272: 31441-31446Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The systematic exchange of single guest triplets embedded in an otherwise constant environment allows their influence on folding to be related to their contribution to triple-helix stability. The relationship between folding half-times and the melting temperatures of the host-guest peptide concentrations set at 1 mg/ml was considered (Fig. 5). All Gly-Xaa-Hyp peptides have fast folding rates and high stabilities, with a small range for botht 12 values and melting temperature values. The Gly-Pro-Yaa peptides show a broad range for both folding half-times (12–98 min) and melting temperatures (30–45 °C), with the more stable peptides tending to fold faster. For Gly-Xaa-Yaa triplets with no imino acids, four peptides with similar thermal stabilities were found to have very different folding times. This suggests the interactions determining folding differ from those important for stability for tripeptides with no imino acids. The host-guest peptide set shows the wide range in effectiveness of Gly-Xaa-Yaa tripeptides in a fixed Gly-Pro-Hyp environment to facilitate or depress nucleation. The nucleation step of peptides differs from that of collagen in that this step occurs in three independent peptides, whereas triple-helix nucleation in collagen occurs in a molecule that is linked together by the association of disulfide-linked C-propeptides (Fig. 4). Despite this difference, it is likely that the propensity of individual tripeptides to nucleate a peptide triple helix can be applied to the ability of different sequences at the C terminus of collagen to serve as a nucleation site. In addition, it is possible that interruptions in the (Gly-Xaa-Yaa)n-repeating sequence, as found normally in basement membrane collagen or for osteogenesis imperfecta Gly→Xaa mutations in type I collagen, may terminate propagation (38Bächinger H.P. Morris N.P. Davis J.M. Am. J. Med. Genet. 1993; 45: 152-162Crossref PubMed Scopus (114) Google Scholar, 39Liu X. Kim S. Dai Q.-H. Brodsky B. Baum J. Boichemistry. 1998; 37: 15528-15533Crossref PubMed Scopus (55) Google Scholar), making a renucleation event necessary to complete triple-helix formation. Information on the propensity of different Gly-Xaa-Yaa triplets to promote nucleation will aid in the identification of such renucleation sequences. We are grateful to Alan Kirkpatrick for peptide synthesis and Nick Bartone for peptide characterization. We also thank Drs. Naina K. Shah, Wei Yang, and Ellen Doss for helpful comments, Chirag Shah for preliminary results, and Ms. Virginia C. Chan for experimental guidance in the early stages of this project. Furthermore, we appreciate the continuous discussions with Dr. Jean Baum.
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