Synthesis and physical properties of (hydroxyproline-proline-glycine)10: Hydroxyproline in the X-position decreases the melting temperature of the collagen triple helix

The collagen-like polytripeptide (hydroxyproline-proline-glycine)₁₀ was synthesized with a solid-phase procedure. Analytical ultracentrifugation indicated that the peptide in aqueous solution at 6 °C had a molecular weight of 2550, the expected size of a single chain. The peptide had a relatively small negative optical rotation at 578 nm, and it did not show a thermal transition as is seen with collagen or collagen-like polytripeptides which form triple helices. At low temperatures in aqueous solution, the circular dichroism spectrum was similar to that of triple-helical collagen and collagen-like peptides in that there was a positive peak at 224 nm and a negative peak at 200 nm. The amplitudes of the peaks, however, were considerably less than the peaks obtained with triple-helix proteins and peptides. Since (proline-proline-glycine)₁₀ was triple helical under the same conditions, the results demonstrated that hydroxyproline in the X-position of the repeating -glycine-X-Y- sequences decreases rather than increases, the thermal stability of the triple helix. This positional specificity cannot be explained by any of the current models for the structure of the triple helix or any of the current proposals for how hydroxyproline stabilizes the structure.     

Triple helix–coil transition of covalently bridged collagenlike peptides

The thermal triple helix–coil transition of covalently bridged collagenlike peptides with repeating sequences of (Ala-Gly-Pro)_n, n = 5–15, was studied optically. The peptides were soluble in water/acetic acid (99:1) and were found to form triple-helical structures in this solvent system beginning with n = 8. The thermodynamic analysis of the transition equilibrium curves for n = 9–13 yielded the parameters ΔH°_s = ?7.0 kJ per tripeptide unit, ΔS°_s = ?23.1 J deg^?1 mol^?1 per tripeptide unit for the coil-to-helix transition, and the apparent nucleation parameter σ ? 5 × 10^?2. It was suggested through double-jump temperature experiments that the rate-limiting step during refolding is not only influenced by the difficulties of nucleation, but also by cis–trans isomerization of the Gly-Pro peptide bond.     

Conformational change of the triple-helical structure. II. Conformation of (Pro-Pro-Gly)n and (Pro-Pro-Gly)n (Ala-Pro-Gly)m(Pro-Pro-Pro-Gly)n in an aqueous solution

Measurements of the molecular weight of (Pro-Pro-Gly)_n and (Pro-Pro-Gly)_n(Ala-Pro-Gly)_m(Pro-Pro-Gly)_n, which were synthesized by the solid-phase method, revealed that they formed a trimer in an aqueous solution, and dissociated into single-stranded chains on warming. Accompanying the transition, a large decrease of optical rotation was observed, like the collagen–gelatin transition. The shape of the trimeric molecule was rodlike, and the dimensions were 12 Å in diameter and 2.8 Å per residue in length, regardless of the length of Ala-Pro-Gly sequences in a peptide chain. The data indicate that both Pro-Pro-Gly sequences and Ala-Pro-Gly sequences from the triple-helical structure similar to that of collagen in aqueous solution. All optical rotational dispersion (ORD) curves of solutions of the peptides were represented by a single-term Drude equation, and the Drude constant λ_c was 200 nm for all peptides regardless of the length of Ala-Pro-Gly sequences. The resemblance between the helical structure formed by Pro-Pro-Gly sequences and that by Ala-Pro-Gly sequences was also suggested by the formation of the hybrid triple helix from two kinds of peptide chains with different lengths of Ala-Pro-Gly sequences.     

Conformational analysis of polytripeptides (Gly-Pro-Ala)n, (Gly-Ala-Hyp)n,and (Gly-Ala-Ala)n in connection with the problem of collagen structure

Conformational analysis of triple helics of a type of collagen was performed with typical collagen tripeptide sequences based on Gly-Pro-Ala, Gly-Ala-Hyp, and Gly-Ala-Ala. During energy minimization, the possibility of continual deformation of the pyrrolidine cycle was taken into account in order to achieve better accuracy in the resulting structure. The (Gly-Pro-Ala)_n structure is almost isomorphic to the (Gly-Pro-Hyp)_n structure obtained in the previous work [Tumanyan, V. G. & Esipova, N.G. (1982) Biopolymers 21 , 475–497]. For a collagen-type structure, the optimal conformation of (Gly-Ala-Hyp)_n tends to have a decreased unit twist (t = 15°), although the energy advantage with respect to the conformation with t = 45° is not so significant. A similar situation is observed for (Gly-Ala-Ala)_n. In this case, the energy decrease during unwinding to t = 15° from t = 45° is quite small. The conformations of (Gly-Ala-Hyp)_n and (Gly-Ala-Ala)_n with t = 15° exhibit a similarity with a triple complex of polyproline II helices—a noncoiled coil such as (Gly-Pro-Hyp)_n and (Gly-Pro-Ala)_n. A similar structure may be postulated for subcomponent cq1 of the first component of a human complement containing substantial Gly-X-Pro and Gly-X-Y tripeptide derivatives in the primary structure (X, Y = any amino acid). The results suggest that the observed helical symmetry of collagen (t = 36°) is a consequence of superposition of diffraction patterns (for sufficiently long segments) from various helices (t varies from ～15° for Gly-X-Hyp and Gly-X-Y to ～56° for Gly-Pro-Ala). For short alternating segments, some unification of different helical structures is possible.     

Polypeptide models of collagen: Properties of (Pro-Pro-betaAla)n.

We have synthesized (Pro-Pro-βAla)_n as a model for collagen. The synthetic polytripeptide, mol wt 6500, exhibits a large negative optical rotation with a very strong negative Cotton effect centered at 216 nm. The optical rotatory dispersion of (Pro-Pro-βAla)_n followed a single-term Drude equation and the λ_c was 195 nm. The rotation decreased markedly on heating with the midpoint of the broad transition at 55°C. Preliminary studies also showed loss of structure in guadinine HCl. The circular dichroism spectrum of the polymer exhibited a deep trough at 190 nm. The marked similarities of solution properties of (Pro-Pro-βAla)_n to (Pro-Pro-Gly)_n suggest that β-alanine can replace glycine in generating collagen-like helix in solution.     

Determination of the rates and barriers to conformational isomerization in the dipeptide L-pro-L-4hyp by direct 13C nmr thermal equilibration

The ¹³C nmr equilibration method lends itself as a tool for study of conformational rate processes involving aqueous media in conjunction with high activation barriers. This method is applied for measurement of kinetic and thermodynamic parameters of isomerism in the dipeptide L -Pro-L -4Hyp. The activation barrier for cis ? trans interconversion (ω 0° → 180°) is determined, ΔG^≠ = 22.3 kcal/mol. From low-temperature study, an upper limit ΔG^≠ < 9.7 kcal/mol is evaluated for cis′ ? trans′ rotation (ψ ?40° → 160°). These data are compared with computed values found in literature. The results are discussed in connection with the helix–coil transition of collagen involving Gly-L -Pro-L -4Hyp as nucleation sites.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

Helix-coil transition of Poly(L-glutamic acid) in N-methylacetamide

The folding of randomly coiled poly(L -glutamic acid) to the helical state has been studied in N-methylacetamide by titration methods. Since this solvent would be expected to form amide-peptide group hydrogen bonds with the unfolded form of the polymer, to a first approximation no helix stabilization could come from intrapolymer hydrogen bonds. The titration data, collected from 30 to 70°C yield the following values per residue for the thermodynamic parameters governing the coil-helix reaction for the uncharged polymer: ΔG_30°C°, ?1. 9 ± 0.1 kcal; Δ H°, 0 ± 0.1 kcal; ΔS_30°C°, 6.3 ± 0.6 eu. In N-methyl acetamide, the helix is an order of magnitude more stable than in water, and this stabilization appears to be entirely the result of the entropy gained by solvent molecules which are released from the polymer upon folding.     

Collagen-based structures containing the peptoid residue N-isobutylglycine (Nleu): Synthesis and biophysical studies of Gly-Pro-Nleu sequences by circular dichroism,ultraviolet absorbance,and optical rotation

A peptoid residue N-isobutylglycine (Nleu) was introduced as a proline surrogate in collagen-like triple helical structures. A series of single chain and template-assembled collagen-based peptide-peptoid structures composed of Gly-Pro-Nleu sequences were prepared by solid-phase segment condensation methods. Both a synthetic route in solution and a solid phase method were employed to couple the KTA (cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid, also known as the Kemp triacid) based template, KTA-(Gly-OH)₃, to peptide-peptoid chains. Biophysical studies using CD, uv absorbance, and optical rotation measurements demonstrated that these compounds form triple-helical structures when the chains are longer than critical lengths. Results from melting curve measurements indicated that the Gly-Pro-Nleu sequence is comparable to the Gly-Pro-Pro sequence in stabilizing a triple-helical conformation. The KTA-based template stabilized triple-helical structures as can be seen by the increased melting temperatures as compared to equivalent single chain molecules. In addition, the template reduced the minimum chain length necessary to form a triple helix from six to only three trimer repeats. © 1996 John Wiley & Sons, Inc.     

Polypeptide models of collagen. II. Solution properties of (Pro-Gly-Phe)n

The conformation of (Pro-Gly-Phe)_n in trifluoroethanol was investigated using CD, nmr and ir techniques. After making appropriate correction for the contribution of the phenylalanine chromophore to the observed CD spectra of the polytripeptide at several temperatures, it is found that (Pro-Gly-Phe)_n can exist in a partially triple-helical conformation in this solvent a t low temperatures. The nmr and ir data support this conclusion. In conjunction with recent theoretical sutdies, our data offer an explanation for the preferential occurrence of the Phe residue in position 2 of the tripeptide sequence Gly-R₂-R₃, in collagen.     

The formation of the triple helix in a mixed solution of (Pro-Pro-Gly)n and (Pro-Pro-Gly)n'. A model of molecular size recognition

A theoretical analysis is given of the triple-helix–random-coil transition in a mixed solution of poly(Pro-Pro-Gly)_n with two different but defined degrees of polymerization n and n′. Because of the highly cooperative nature of this helix–coil transition, each polypeptide chain tends to form a triple helix with other polypeptide chains with the same degree of polymerization (size recognition). Occurrence of triple helices consisting of polypeptide chains with different degrees of polymerization (error in recognition) is studied in detail as a function of the cooperativity, and n and n′. Implication of this analysis for molecular recognition in general is discussed.     

Hydration dynamics of the collagen triple helix by NMR

The hydration of the collagen-like Ac-(Gly-Pro-Hyp)(6)-NH(2) triple-helical peptide in solution was investigated using an integrated set of high-resolution NMR hydration experiments, including different recently developed exchange-network editing methods. This approach was designed to explore the hydration dynamics in the proximity of labile groups, such as the hydroxyproline hydroxyl group, and revealed that the first shell of hydration in collagen-like triple helices is kinetically labile with upper limits for water molecule residence times in the nanosecond to sub-nanosecond range. This result is consistent with a "hopping" hydration model in which solvent molecules are exchanged in and out of solvation sites at a rate that is not directly correlated to the degree of site localization. The hopping model thus reconciles the dynamic view of hydration revealed by NMR with the previously suggested partially ordered semi-clathrate-like cylinder of hydration. In addition, the nanosecond to sub-nanosecond upper limits for water molecule residence times imply that hydration-dehydration events are not likely to be the rate-limiting step for triple helix self-recognition, complementing previous investigations on water dynamics in collagen fibers. This study has also revealed labile proton features expected to facilitate the characterization of the structure and folding of triple helices in collagen peptides.     

Stabilization mechanism of triple helical structure of collagen molecules

Summary The role of 4-hydroxyproline (Hyp) in stabilizing collagen triple helical structure has been investigated comprehensively. Recently it was emphasized that the preferential pyrrolidine ring pucker influenced by the stereoelectronic effects of substituted groups mainly affects the thermal stability of the triple helix. To examine this explanation, we synthesized and characterized (fPro ^R -Pro-Gly)₁₀ and (fPro ^S -Pro-Gly)₁₀. According to the results of CD and analytical ultracentrifugation, (fPro ^S -Pro-Gly)₁₀ takes a triple helical structure and (fPro ^R -Pro-Gly)₁₀ exists in a single chain structure, the trend of which is not consistent with the relationship between (Hyp ^S -Pro-Gly)₁₀ and (Hyp ^R -Pro-Gly)₁₀. In order to rationalize experimental results as a whole, we carried out DSC analyses and determined the thermodynamic parameters associated with the structural transition of these collagen model peptides. In this paper, we reported the DSC results for (Pro-Pro-Gly)₁₀, (Pro-Hyp ^R -Gly)₁₀ and (Pro-fPro ^R -Gly)₁₀ as a part of this study. Based on those parameters, we concluded that Hyp and fPro stabilize the triple helix in different stabilizing mechanisms; the increased stability of (Pro-Hyp ^R -Gly)₁₀ is ascribed primarily to the enthalpic effects while that of (Pro-fPro ^R -Gly)₁₀ is achieved through the entropic ones.     

The effect of deuterium oxide on the stability of the collagen model peptides H‐(Pro‐Pro‐Gly)10‐OH,H‐(Gly‐Pro‐4(R)Hyp)9‐OH,and Type I collagen

The collagen triple helix has a larger accessible surface area per molecular mass than globular proteins, and therefore potentially more water interaction sites. The effect of deuterium oxide on the stability of collagen model peptides and Type I collagen molecules was analyzed by circular dichroism and differential scanning calorimetry. The transition temperatures (T_m) of the protonated peptide (Pro‐Pro‐Gly)₁₀ were 25.4 and 28.7°C in H₂O and D₂O, respectively. The increase of the T_m of (Pro‐Pro‐Gly)₁₀ measured calorimetrically at 1.0°C min^?1 in a low pH solution from the protonated to the deuterated solvent was 5.1°C. The increases of the T_m for (Gly‐Pro‐4(R)Hyp)₉ and pepsin‐extracted Type I collagen were measured as 4.2 and 2.2°C, respectively. These results indicated that the increase in the T_m in the presence of D₂O is comparable to that of globular proteins, and much less than reported previously for collagen model peptides [Gough and Bhatnagar, J Biomol Struct Dyn 1999, 17, 481–491]. These experimental results suggest that the interaction of water molecules with collagen is similar to the interaction of water with globular proteins, when the ratio of collagen to water is very small and collagen is monomerically dispersed in the solvent. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 93–101, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The role of water in the crystal structure of N-acetyl-L-4-hydroxyproline

The crystal structure of N-acetyl-L -4-hydroxyproline (Hyp) was determined by direct methods. (The crystal is orthorhombic with the space group P2₁2₁2₁.) The acetyl group is in the trans conformation and the pyrrolidine ring puckers at C^γ (C^sC^γ envelope), as in most Hyp residues. According to the rotation angle ψ = ?30°, the N-acetyl-L -4Hyp has the same conformation as an α-helix of prolyl residues. The crystal packing is stabilized by hydrogen bonds between three different molecules and the same molecule of water. One of the water bridges involves the carbonyl of the N-acetyl group of one molecule and the hydrogen atom of the 4-OH group of another. Such an arrangement has been proposed to explain the high stability of (Gly-L -Pro-L -4Hyp)_n. A second bridge involves the two hydrogens of the water molecule and the carbonyl groups of two neighbouring molecules, as already proposed in a dihydrated model of collagen. These experimental features, which are discussed in relation to the different models of collagen, allow us to propose an hypothetical arrangement for the water molecule which is strongly retained in the triple helix of (Gly-L -Pro-L -4Hyp)_n.     

A triple-helical model for (Gly-Pro-Hyp)n with cis peptide units

Synthetic regular polytripeptides of the type (Gly-R₂-R₃) where R₂, R₃, or both, are imino acids have been widely studied as model compounds for collagen. One such polytripeptide is poly(Gly-Pro-Hyp), since triplets with this sequence constitute about 10% of collagen. Recently, a new model has been proposed for this polytripeptide in which one of the three peptide bonds in the tripeptide unit is in the cis conformation, and the γ-hydroxyl group of hydroxyproline forms a direct interchain hydrogen bond within the triple helix. We have confirmed this structure by model building using computer techniques, and the helical parameters obtained by us are close to the experimentally observed values. The model is also found to be comparable in stability with other models from energy considerations.     

Conformational change of the triple-helical structure. III. Stabilizing forces in the triple helix

The analysis of thermal melting curves of (PPG)_n (n = 10, 12, 14, and 15) and (PPG)_n(APG)_m (PPG)_n (2n + m = 15; m = 1, 3, and 5) revealed that the enthalpy and entropy changes accompanying the transition from the random coil to the triple helix are ?2500 cal and ?6.3 e.u. per one mole of the tripeptide of the form of Pro-Pro-Gly, and ?3100 cal and ?11.2 e.u. per one mole of the tripeptide of the form of Ala-Pro-Gly. The thermal instability of the triple helix composed of Ala-Pro-Gly sequences, compared to the helix of Pro-Pro-Gly sequences, is due to the larger entropy change of Ala-Pro-Gly (?11.2 e.u.) compared to that of Pro-Pro-Gly (?6.3 e.u.), not from the difference in the enthalpy change. The difference in the enthalpy change between Pro-Pro-Gly and Ala-Pro-Gly arises from the hydrophobic bond between two pyrrolidine rings of proline residues formed in the triple helix. Since the enthalpy change for the formation of hydrophobic bonds is positive, it is also concluded that only one hydrogen bond is formed in a tripeptide unit, regardless of the amino acid sequence. The enthalpy change for the formation of this hydrogen bond is ?3100 cal/mol, and that of the hydrophobic bond between two pyrrolidine rings is +600 cal/mol.     

Polypeptide models of collagen. Solution properties of (Gly-Pro-Sar)n and (Gly-Sar-Pro)n.

Our studies on the solution conformation of (Gly-Pro-Sar)_n and (Gly-Sar-Pro)_n synthesized as polypeptide models for collagen are reported. It is found that, while (Gly-Pro-Sar)_n exists in ordered triple-helical conformation, (Gly-Sar-Pro)_n remains as a disordered random coil in water. Addition of certain helix-promoting solvents seems to generate order in (Gly-Sar-Pro)_n.     

The Contribution of Interchain Salt Bridges to Triple-Helical Stability in Collagen

Studies on collagen and collagen-like peptides suggest that triple-helical stability can vary along the amino acid chain. In this regard, it has been shown that lysine residues in the Y position and acidic residues in the X′ position of (GPO)₃GXYGX′Y′(GPO)₃ peptides lead to triple-helical structures with melting temperatures similar to (GPO)₈ (where O is hydroxyproline), which is generally regarded as the most stable collagen-like sequence of this length. This enhanced stability has been attributed to the formation of salt bridges between adjacent collagen chains. In this study, we explore the relationship between interchain salt bridge formation and triple-helical stability using detailed molecular simulations. Although our results confirm that salt bridges promote triple-helical stability, we find that not all salt bridges are created equal. In particular, lysine-glutamate salt bridges are most stabilizing when formed between residues in the middle strand (B) and the trailing strand (C), whereas lysine-aspartate salt bridges are most stabilizing when formed between residues in the leading (A) and middle (B) strand—the latter observation being consistent with recent NMR data on a heterotrimeric model peptide. Overall, we believe these data clarify the role of salt bridges in modulating triple-helical stability and can be used to guide the design of collagen-like peptides that have specific interchain interactions.     

Characterization of triple-helical conformations and melting analyses of synthetic collagen-like peptides by reversed-phase HPLC

There is a confusion in the application of circular dichroism (CD) spectroscopy in analyzing collagen's structure for the overlapping of the spectral shapes and positions of the collagen triple helix and poly(proline-II)-like structure. The unique repetitive sequence of the collagen triple helix is susceptible to misalignment during the spontaneous assembly. Such misaligned structures are usually difficult to be characterized by CD or NMR spectroscopy. Here, RP-HPLC was developed as a conformational characterization technique for synthetic collagen-like peptides based on the different hydrophobicities exhibited by the triple-helical and unassembled peptides. RP-HPLC was also used to study thermal transitions and to measure melting point temperatures (Tm) of the collagen-like peptides.