INTRODUCTION

The triple-helix conformation is a major protein motif found in the collagen family and in a number of host-defense proteins (1). The collagen triple-helix is composed of three polypeptide chains, each in a polyproline II-like helix, that are folded around each other in a supercoiled rod-like structure (2-4). A consequence of this folding pattern is that only Gly is small enough to fit as every third residue in each polypeptide chain where the three chains pack in close proximity. This steric constraint generates the repeating sequence pattern (Gly-X-Y)_n, where proline (Pro) is frequently found in the X-position while hydroxyproline (Hyp)¹ is frequently found in the Y-position. Studies on collagens and peptides show that the sequence Gly-Pro-Hyp is the most stabilizing tripeptide unit for the triple-helix conformation (5-7). Imino acid residues favor triple-helix formation entropically because their  and  angles are sterically constrained close to the values found in collagen chains. Hyp, formed by post-translational hydroxylation of prolines in the Y-position, provides a stability greater than Pro residues in native collagens and peptides (5, 8). A highly stereospecific effect of Hyp in the Y-position is indicated by the dramatic destabilization when Hyp is in the X- rather than the Y-position, when 4-Hyp is replaced by 3-Hyp, or when the hydroxyl group is placed in the opposite orientation with respect to the pyrrolidine ring (cis-Hyp) (9, 10). Thermodynamic investigations indicated that the additional stabilization by Hyp is a result of hydrogen bonding (11), and a recently solved crystal structure showed its pivotal role in a highly ordered, water-mediated hydrogen bonding network (4, 12).

Because of its stabilizing nature, Gly-Pro-Hyp tripeptides were used as a basis for a host peptide sequence, within which a guest triplet is introduced into a common stabilizing framework (13). Host-guest peptides have previously been shown to be a valuable tool for understanding the propensity of different amino acids for -helical (14, 15) and -sheet structures (16). A stable triple-helix conformation was adopted by host-guest peptides of the design Ac(Gly-Pro-Hyp)₃-Gly-X-Y-(Gly-Pro-Hyp)₄-Gly-Gly-NH₂, and the stability varied with the specific Gly-X-Y triplet introduced in the guest position. Incorporation of the most common non-polar residues in collagen (Ala, Leu, Phe) in the X- or Y-positions of the guest triplet decreased the thermal stability relative to the host (Gly-Pro-Hyp)₈ peptide (13). Recent studies have also been carried out on host-guest peptides incorporating individual charged residues or pairs of oppositely charged residues in the X- and Y-positions (27). An unexpected observation was that the Gly-Pro-Arg host-guest peptide showed a stability comparable to the Gly-Pro-Hyp-containing host peptide, a stability greater than seen for all other charged, as well as uncharged, triplets (13, 17).

The surprisingly high stability of the host-guest peptide with a Gly-Pro-Arg guest triplet prompted further investigations to clarify the interactions of arginine in the triple-helical structure. Peptides related to the Gly-Pro-Arg host-guest peptide were designed to evaluate the critical features in the Arg side chain and to investigate whether the high stability is conferred by multiple Gly-Pro-Arg triplets.

MATERIALS AND METHODS

Peptides were synthesized on an Applied Biosystems 430A Synthesizer using the standard FastMoc (Applied Biosystems) method on Fmoc-RINK (N-(9-fluorenyl)methoxycarbonyl-RINK) resin. Side chain protection groups used were t-butyl for Hyp, benzyloxycarbonyl for Lys, and pentamethylchroman-sulfonyl for Arg. The N and C termini were blocked by acetylation and amidation, respectively, to increase stability (18) and eliminate charges at locations other than the guest triplet. A host-guest peptide containing homoArg was synthesized from the peptide containing Gly-Pro-Lys guest triplet by reaction of the peptide (5% in water) with 0.5 M 1-guanyl-3,5-dimethylpyrazole nitrate (Aldrich) at pH 9.0 for 8 days at 20 °C (19). Peptides were purified by reversed phase HPLC³ on a Shimadzu HPLC system using a YMC C-18 column (20 × 250 mm). Composition and identity of the peptides were confirmed by amino acid analysis, using a Waters HPLC system with ninhydrin detection, and by laser desorption mass spectrometry using a Brucker MALDI-TOF instrument.

CD spectra were recorded in quartz cells of 1-mm path length on an Aviv model 62DS spectropolarimeter equipped with a Hewlett-Packard Peltier thermoelectric temperature controller. Peptides were dried in vacuo over P₂O₅ for 48 h prior to weighing to prepare solutions of 1 mg/ml. Generally, peptides were dissolved in PBS (10 mM sodium phosphate and 0.15 M NaCl, pH 7.0) and were equilibrated at 4 °C for at least 48 h before CD measurements. Other buffers used were 0.1 M Tris/HCl, pH 7.0, or 0.1 M acetic acid, pH 2.7. Thermal stability measurements were carried out as described previously (13). Transition curves were normalized and corrected for monomer and trimer contributions (5, 13, 17, 18). Melting temperatures (T_m) were taken at a fraction folded of 0.5.

Peptides (c = 1 mg/ml in PBS) were denatured at 70 °C for 15 min and were then rapidly cooled by quenching in an ice-salt bath at about 6 °C prior to transfer into a 1-mm CD cell precooled to 5 °C, as described previously (20). Ellipticity at 225 nm was followed for 3 h.

RESULTS AND DISCUSSION

The CD spectrum of the Gly-Pro-Arg host-guest peptide (GPR) showed the characteristics of a triple-helix with a maximum at 225 nm at 2 °C (in PBS, pH 7.0), and heating resulted in a sharp transition with a T_m of 45.5 °C (Fig. 1; Table I). This T_m value is the same as observed for the most stable (Gly-Pro-Hyp)₈ host peptide (GPO) under identical conditions (Table I). This indicates that in the Gly-Pro-Hyp-rich environment of the host peptide, Gly-Pro-Arg has a stabilizing influence as great as that of Gly-Pro-Hyp. The T_m value of 45.5 °C observed for GPO and GPR peptides represents the maximum stability observed for the host-guest peptides studied thus far.

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Table I. Peptide sequences and their melting temperatures Tm Measurements were performed in PBS, pH 7.0, except for peptide GPR8. Peptide Sequence Tm °C GPO Ac(Gly-Pro-Hyp)8-Gly-Gly-NH2 45.5a GPR Ac(Gly-Pro-Hyp)3-Gly-Pro-Arg-(Gly-Pro-Hyp)4-Gly-Gly-NH2 45.5 GRO Ac(Gly-Pro-Hyp)3-Gly-Arg-Hyp-(Gly-Pro-Hyp)4-Gly-Gly-NH2 40.6 GPK Ac(Gly-Pro-Hyp)3-Gly-Pro-Lys-(Gly-Pro-Hyp)4-Gly-Gly-NH2 36.8 GPhR Ac(Gly-Pro-Hyp)3-Gly-Pro-homoArg-(Gly-Pro-Hyp)4-Gly-Gly-NH2 42.8 GAO Ac(Gly-Pro-Hyp)3-Gly-Ala-Hyp-(Gly-Pro-Hyp)4-Gly-Gly-NH2 39.9a GAR Ac(Gly-Pro-Hyp)3-Gly-Ala-Arg-(Gly-Pro-Hyp)4-Gly-Gly-NH2 38.2 GAK Ac(Gly-Pro-Hyp)3-Gly-Ala-Lys-(Gly-Pro-Hyp)4-Gly-Gly-NH2 30.8 RR Ac(Gly-Pro-Hyp)4-Gly-Pro-Arg-Gly-Pro-Arg-(Gly-Pro-Hyp)2-Gly-Gly-NH2 40.4 ROR Ac(Gly-Pro-Hyp)3-Gly-Pro-Arg-Gly-Pro-Hyp-Gly-Pro-Arg-(Gly-Pro-Hyp)2-Gly-Gly-NH2 42.2 ROOR Ac(Gly-Pro-Hyp)2-Gly-Pro-Arg-(Gly-Pro-Hyp)2-Gly-Pro-Arg-(Gly-Pro-Hyp)2-Gly-Gly-NH2 42.8 GPR8 Ac(Gly-Pro-Arg)8-Gly-Gly-NH2 32.6b a In agreement within experimental error limits with that previously reported (13). b Measured in 2 M NaCl, 10 mM sodium phosphate, pH 7.0.

To investigate whether the stability is related to the presence of phosphate in PBS, the melting curve for GPR was also examined in acetic acid at pH 2.7 and Tris/HCl at pH 7.0. In both these systems, the T_m value remained the same, indicating that the unexpectedly high T_m for GPR occurs in different buffers and at acidic as well as neutral pH.

Additional host-guest peptides were designed to dissect the contributions of charge, side chain functional group, and length of the side chain to the unusually high stability of the Gly-Pro-Arg peptide. Peptide GPK (Table I), which also has a positively charged side chain in the Y-position, was examined under the same set of buffer conditions. This peptide had a T_m of 36.8 °C, almost 8 °C lower than GPR (Fig. 1, Table I) and somewhat less than that of the Gly-Pro-Ala-containing host-guest peptide GPA (T_m = 38.3 °C) (13), indicating that a positive charge in the Y-position is not by itself sufficient to confer the maximal stability for this peptide set.

To explore the influence of the side chain length of the arginine and thus the spatial specificity of the guanidino group relative to the peptide backbone, a peptide incorporating homoArg (GPhR) was studied (Table I). The side chain of homoArg has a guanidino group but is 1 CH₂ unit longer than that of Arg. The T_m of GPhR was 42.8 °C. The value is somewhat lower than seen for GPR, and it is possible that increased mobility of the lengthened side chain leads to a 3 °C decrease in stability. The T_m of GPhR is 6 °C higher than GPK and 5 °C higher than GPA, indicating that the guanidino group in homoArg is still conferring a positive stabilizing influence on the triple-helix.

Host-guest peptides discussed thus far are of the form Gly-Pro-Y. To investigate whether the proline preceding arginine is a determining factor in the high stability of GPR, a series of 3 additional host-guest peptides with Gly-Ala-Y guest triplets were examined. The T_m values were 39.9 °C for GAO, 38.2 °C for GAR, and 30.8 °C for GAK (Fig. 1, Table I). These results follow the general trends seen for Gly-Pro-Y guest triplets, suggesting that the stabilizing effect of Arg in the Y-position also occurs when residues other than a proline are in the X-position.

In the highly stable GPR peptide, Arg is present in the Y-position of the Gly-X-Y guest triplet. To investigate whether the stabilizing effect of Arg occurs in the X- as well as in the Y-position, peptide GRO was examined (Table I). The T_m of GRO is 40.6 °C, about 5 °C lower than GPR (Fig. 1, Table I). This indicates that Arg is more stabilizing in the Y-position than in the X-position of the guest triplet, even though GRO contains the stabilizing Hyp residue. This contrasts with all other guest triplets studied so far, where placement of a non-polar or ionizable residue in the Y-position is more destabilizing than in the X-position. For example, the host-guest peptide Gly-Pro-Leu is less stable than Gly-Leu-Hyp (13), and GPK is less stable than GKO (27). The preference of non-polar residues for the X-position has been explained in part on the basis of steric hindrance of the Y-position residue with the neighboring chain (13, 21). The only residue besides Arg which is more favorable in the Y-position than in the X-position is Hyp. The peptide (Hyp-Pro-Gly)₁₀ with Hyp in the X-position is unstable, while (Pro-Hyp-Gly)₁₀ with Hyp in the Y-position has T_m of 58 °C (10). This preference of arginine and hydroxyproline for the Y-position suggests a similarity in their stabilizing effect.

It has been shown that clustering of Gly-Pro-Hyp triplets magnifies its stabilizing effect (7). To further test the equivalence between Gly-Pro-Arg and Gly-Pro-Hyp triplets, the peptide (Gly-Pro-Arg)₈ (GPR8) was compared with the host peptide (Gly-Pro-Hyp)₈ (GPO). In contrast to the high triple-helix content and stability of GPO, GPR8 showed little sign of triple-helix in its CD spectrum after 48 h at 4 °C in PBS. However, after incubating GPR8 at 4 °C for 28 days, the ellipticity ([]₂₂₅ = +1300 degrees·cm²·dmol¹) indicated the presence of about 30% triple-helix content. Increasing GPR8 concentration to 3 mg/ml resulted in an almost completely triple-helical structure after 28 days at 4 °C. In the presence of 2 M NaCl, the peptide reached a fully triple-helical conformation within 14 days, and this refolded sample had a T_m of 32.6 °C (Table I).

The inability of GPR8 to form a triple-helix in PBS at a concentration of 1 mg/ml and its salt dependence suggests that a high density of positive charge may hinder triple-helix formation. To better define such charge repulsion, two Gly-Pro-Arg triplets were integrated into the host-guest peptide set. The two Gly-Pro-Arg triplets were separated by either zero (RR), one (ROR), or two Gly-Pro-Hyp triplets (ROOR) (Table I). Peptide RR had a T_m of 40.4 °C, a reduction of about 5 °C compared with GPR (Table I, Fig. 2). This is likely due in part to charge repulsion between neighboring charged triplets. Separation of the two Gly-Pro-Arg triplets by either one or two Gly-Pro-Hyp triplets, in ROR or ROOR, respectively, increased the stability to near 42 °C (Table I, Fig. 2), suggesting that charge repulsion is eliminated with one or more intervening Gly-Pro-Hyp tripeptides. Regardless of their separation, the thermal stability of peptides with two Gly-Pro-Arg triplets stayed lower than that with single Gly-Pro-Arg triplet. Although this might result from the presence of two Gly-Pro-Arg triplets, it can also reflect a limitation of the peptide design, since the placement of the second Gly-Pro-Arg triplet at a different position with respect to the termini could affect its overall stability.

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In addition to its stabilizing influence, hydroxyproline residues have been shown to accelerate the folding of the triple-helix (23).⁴ The folding of GPO was faster than for all other host-guest peptides studied and did not fit well to first, second, or third order kinetics.⁵ Even though GPR has the same maximal stability as GPO, it shows a slower folding and fits second order kinetics (Fig. 3). The folding rates of GPR-related peptides were compared, and all fit second order kinetics, in the following order: GPR  GPhR > GPK > RR  ROR  ROOR (Fig. 4), whereas the order of relative stabilities is: GPR > GPhR  ROR = ROOR > RR > GPK. This indicates that there is no simple relationship between stability and folding, and different features must be involved in these two properties. The data suggest that charge repulsion delays folding, which would explain the slower folding of GPR, where the positively charged Arg from 3 chains must come together, compared with GPO. This is consistent with the slow folding rate seen for all three peptides with two Gly-Pro-Arg triplets and also supported by the extremely slow folding seen for GPR8 and the accelerated folding observed in the presence of high ionic strength (Fig. 3).

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The results indicate stabilization of the triple-helix when Arg is in the Y-position of a Gly-X-Y triplet. The Arg stabilization must relate to the nature of the guanidino group of Arg and its interaction with the peptide backbone and/or with the water network in the triple-helix. Arg is unique in its hydrogen bonding capacity, with up to five potential bonding sites (24). Computational modeling (SYBYL 6.2) shows that the guanidino group in the Gly-Pro-Arg sequence of the host-guest peptide can participate in direct hydrogen bonding to the peptide backbone carbonyl groups within the same chain or to the Arg carbonyl group of the neighboring chain (Fig. 4), in agreement with previous reports (25). When one NH is hydrogen-bonded to a backbone carbonyl, additional constraints are conferred on the remaining hydrogen bonding sites of the guanidino group, and these could participate in the ordered hydration network of the triple-helix. However, similar hydrogen bonding schemes are possible for peptides GPK, GPhR, and GRO, and in fact, their enthalpy values are as great or greater than that of peptide GPR (27) (data not shown). When Arg is in the Y-position, the limited mobility of its side chain compared with that of Lys and homoArg may restrict the hydrogen bonding to sites favorable for an optimal hydration network, similar to that seen for Hyp.

The inclusion of triplets with Arg in the Y-position provides an alternative to hydroxyproline residues in generating highly stabilized regions of triple-helix. This may explain the high frequency of occurrence of Arg in collagens and the preponderance of Arg residues in the Y-position (26). For example, Gly-X-Arg tripeptide sequences constitute 13% of all triplets in fibril-forming collagens. The high T_m value observed for the host-guest peptide containing a single Gly-Pro-Arg triplet provides a potential strategy for stabilization of triple-helical structures without the need for hydroxylation of Pro residues. This strategy could have application to the production of collagen-like molecules in prokaryotic expression systems, which lack the enzymes for post-translational modification of Pro residues to Hyp (22). The results on peptides indicate a limitation on inclusion of multiple Gly-Pro-Arg triplets, since proximal charged triplets can destabilize the triple-helix, but incorporation of selected Gly-X-Arg triplets could enhance stability.

Collagen function requires formation of a stable triple-helical molecule at body temperature, molecular association to form fibrils or other higher order structures, and the binding to cells and other extracellular matrix components. Gly-Pro-Hyp triplets are known to provide high stability for the triple-helix but do not have the capability for specific interactions. The observation that under certain circumstances Gly-Pro-Arg can confer similar stability as Gly-Pro-Hyp provides a unique example of a tripeptide sequence which is contributing maximally to triple-helix stability and also has the potential for electrostatic interactions in fibril formation and binding.