The effect of a trans-locked Gly-Pro alkene isostere on collagen triple helix stability

An alkene isostere of Gly-trans-Pro was synthesized and incorporated into a host Ac-(Gly-Pro-Hyp)8-Gly-Gly-Tyr-NH2 peptide to investigate the effect of locking a proline amide bond. Proline amide bond isomerization is the slow step in collagen folding. By locking the amide, we hypothesized an increase in stability of the collagen triple helix. The substitution instead destabilized the collagen host peptide. The Tm value of the host control peptide was 50.0 degrees C, while the peptide containing the isostere, Ac-(Gly-Pro-Hyp)3-Gly-psi[(E)CH C]-Pro-Hyp-(Gly-Pro-Hyp)4-Gly-Gly-Tyr-NH2, had a Tm value of 28.3 degrees C. There are clearly factors that contribute to collagen stability and folding that we do not yet understand.     

Inductive Effects on the Energetics of Prolyl Peptide Bond Isomerization: Implications for Collagen Folding and Stability

The hydroxylation of proline residues in collagen enhances the stability of the collagen triple helix. Previous X-ray diffraction analyses had demonstrated that the presence of an electron-withdrawing substituent on the pyrrolidine ring of proline residues has significant structural consequences [Panasik, N., Jr.; Eberhardt, E. S.; Edison, A. S.; Powell, D. R.; Raines, R. T. Int. J. Pept. Protein Res.1994, 44, 262-269]. Here, NMR and FTIR spectroscopy were used to ascertain kinetic and thermodynamic properties of N-acetyl-[β,γ-(13)C]D,L-proline methylester (1); N-acetyl-4(R)-hydroxy-L-proline [(13)C]methylester (2); and N-acetyl-4(R)-fluoro-L-proline methylester (3). The pK(a)'s of the nitrogen atom in the parent amino acids decrease in the order: proline (10.8) > 4(R)-hydroxy-L-proline (9.68) > 4(R)-fluoro-L-proline (9.23). In water or dioxane, amide I vibrational modes decrease in the order: 1 > 2 > 3. At 37 °C in dioxane, the rate constants for amide bond isomerization are greater for 3 than 1. Each of these results is consistent with the traditional picture of amide resonance coupled with an inductive effect that results in a higher bond order in the amide C=O bond and a lower bond order in the amide C-N bond. Further, at 37 °C in water or dioxane equilibrium concentrations of the trans isomer increase in the order: 1 < 2 < 3. Inductive effects may therefore have a significant impact on the folding and stability of collagen, which has a preponderance of hydroxyproline residues, all with peptide bonds in the trans conformation.     

Cobalt-catalyzed chemoselective insertion of alkene into the ortho C-H bond of benzamide

Insertion of 1-alkene, 2-alkene, and styrene into the ortho C-H bond of benzamide in the presence of an inexpensive cobalt catalyst, DMPU as a crucial ligand, and cyclohexylmagnesium chloride proceeds smoothly at 25 °C to selectively give the ortho-alkylated product. Notable features of this reaction include the structural variety of the alkene and the amide substrate and the tolerance of functional groups such as halide, olefin, ester, and amide groups.     

A hyperstable collagen mimic

BACKGROUND: Collagen is the most abundant protein in animals. Each polypeptide chain of collagen is composed of repeats of the sequence: Gly-X-Y, where X and Y are often L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp) residues, respectively. These chains are wound into tight triple helices of great stability. The hydroxyl group of Hyp residues contributes much to this conformational stability. The existing paradigm is that this stability arises from interstrand hydrogen bonds mediated by bridging water molecules. This model was tested using chemical synthesis to replace Hyp residues with 4(R)-fluoro-L-proline (Flp) residues. The fluorine atom in Flp residues does not form hydrogen bonds but does elicit strong inductive effects. RESULTS: Replacing the Hyp residues in collagen with Flp residues greatly increases triple-helical stability. The free energy contributed by the fluorine atom in Flp residues is twice that of the hydroxyl group in Hyp residues. The stability of the Flp-containing triple helix far exceeds that of any untemplated collagen mimic of similar size. CONCLUSIONS: Bridging water molecules contribute little to collagen stability. Rather, collagen stability relies on previously unappreciated inductive effects. Collagen mimics containing fluorine or other appropriate electron-withdrawing substituents could be the basis of new biomaterials for restorative therapies.     

Imino acids and collagen triple helix stability: characterization of collagen-like polypeptides containing Hyp-Hyp-Gly sequence repeats

The analysis of factors contributing to the stability of proteins is a subject of intense debate. Particularly challenging is the study of structural proteins, since their function is their structure. Among these is collagen, the key structural component of bones, skin, cartilage, tendons, and other connecting tissues. It is well established that the collagen triple helix is characterized by the presence of hydroxyproline, whose content modulates triple helix thermal stability according to the requirement of the host organism. Because of the complexity and the fibrous nature of collagen, data on the stability and structure of this protein have been mainly obtained by the use of collagen-like polypeptides. On the basis of CD characterization of collagen-like polypeptides we here show that the presence of Hyp at the X position of repeating triplets Hyp-Hyp-Gly stabilizes the triple helix significantly. This extra-stabilization has been ascribed, by using molecular modeling, to the formation of a hydrogen bond between Hyp residues belonging to the X and the Y positions of adjacent chains. This communication also provides a comprehensive interpretation of the ensemble of available data on polypeptides containing proline derivatives.     

Surprisingly high stability of collagen ABC heterotrimer: evaluation of side chain charge pairs

Type I collagen is a major component of skin, tendon, and ligament and forms more than 90% of bone mass. It is an AAB heterotrimer assembled from two identical alpha1 and one alpha2 chains. However, the majority of studies on the effects of amino acid substitution on triple helix stability have been performed on collagen homotrimeric helices. In a homotrimer, it is impossible to determine whether the contribution to stability is from the polyproline II helix propensity of the amino acids or from interhelix amino acid interactions. The presence of amino acids in all three chains further exaggerates their contribution. In contrast, in a heterotrimer, the individual chains may be tailored in order to have the substitution in one, two, or all three chains. Therefore, a heterotrimer can divulge specific information about any interaction based upon the substitutions in individual chains. In this paper, we evaluate the contribution of electrostatic interactions between side chain charge pairs on the stability of heterotrimers. We synthesize and analyze the stability of four AAB and four ABC heterotrimers including a surprisingly stable ABC heterotrimer composed of (DOG)10, (PKG)10, and (POG)10 chains (O = hydroxyproline). This heterotrimer has a stability comparable to that of a (POG)10 homotrimer even though D and K occur 20 times in the heterotrimeric helix and have been previously shown to significantly destabilize the triple helix compared to the P and O imino acids. These results show that the stability of heterotrimers cannot be directly determined from the analysis of charge pairs in homotrimers. Because collagen heterotrimers can be designed to have substitution in one, two, or three chains, it gives us the ability to decode cross-strand interactions in collagen in a similar fashion to alpha-helical coiled-coil interactions and DNA duplex hydrogen bonding.     

Macrocyclic scaffold for the collagen triple helix

[structure: see text] Three strands of natural collagen are linked by covalent bonds prior to their folding into a triple helix. We report on a synthetic collagen in which the strands are pendent on a rigid macrocyclic scaffold of C(3) symmetry. The scaffold confers substantial conformational stability upon the collagen triple helix and makes its folding independent of concentration, both desirable attributes for exploring and exploiting synthetic collagens.     

Stereoselective synthesis of (Z)-alkene-containing proline dipeptide mimetics

In peptides and proteins, the peptide bond between an amino acid and proline exists as an equilibrium mixture of the cis-imide and trans-imide due to the low energy barrier in their interconversion. This feature greatly influences the structure and function of the proline-containing peptides and proteins. Therefore, restricting the amide bond with an (E)- or (Z)-alkene should provide a promising method for elucidating the structure-activity relationships of the peptide and the proteins. In this report, the regio- and stereoselective synthesis of cis-alanylproline (Ala-Pro) type (Z)-alkene dipeptide mimetic is described. The key steps of this synthesis are to introduce a C3 unit onto a gamma-phosphoryloxy-alpha,beta-unsaturated-delta-lactam with an organocopper-mediated anti-S(N)2' reaction and subsequently construct a five-membered proline-like cyclic structure with an intramolecular Suzuki coupling reaction. Hydrolysis of the amide bond in the resulting bicyclic lactam yields the desired cis-Ala-Pro type (Z)-alkene dipeptide isostere. The presented synthetic methodology should be applicable to the general syntheses of other cis-aminoacylproline type (Z)-alkene dipeptide mimetics.     

Effect of 3-hydroxyproline residues on collagen stability

Collagen is an integral part of many types of connective tissue in animals, especially skin, bones, cartilage, and basement membranes. A fibrous protein, collagen has a triple-helical structure, which is comprised of strands with a repeating Xaa-Yaa-Gly sequence. l-Proline (Pro) and 4(R)-hydroxy-l-proline (4-Hyp) residues occur most often in the Xaa and Yaa positions. The 4-Hyp residue is known to increase markedly the conformational stability of a collagen triple helix. In natural collagen, a 3(S)-hydroxy-l-proline (3-Hyp) residue occurs in the sequence: 3-Hyp-4-Hyp-Gly. Its effect on collagen stability is unknown. Here, two host-guest peptides containing 3-Hyp are synthesized: (Pro-4-Hyp-Gly)(3)-3-Hyp-4-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 1) and (Pro-4-Hyp-Gly)(3)-Pro-3-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 2). The 3-Hyp residues in these two peptides diminish triple-helical stability in comparison to Pro. This destabilization is small when 3-Hyp is in the natural Xaa position (peptide 1). There, the inductive effect of its 3-hydroxyl group diminishes slightly the strength of the interstrand 3-HypC=O.H-NGly hydrogen bond. The destabilization is large when 3-Hyp is in the nonnatural Yaa position (peptide 2). There, its pyrrolidine ring pucker leads to inappropriate mainchain dihedral angles and interstrand steric clashes. Thus, the natural regioisomeric residues 3-Hyp and 4-Hyp have distinct effects on the conformational stability of the collagen triple helix.     

Triple-Helix-Stabilizing Effects in Collagen Model Peptides Containing PPII-Helix-Preorganized Diproline Modules

Collagen model peptides (CMPs) serve as tools for understanding stability and function of the collagen triple helix and have a potential for biomedical applications. In the past, interstrand cross-linking or conformational preconditioning of proline units through stereoelectronic effects have been utilized in the design of stabilized CMPs. To further study the effects determining collagen triple helix stability we investigated a series of CMPs containing synthetic diproline-mimicking modules (ProMs), which were preorganized in a PPII-helix-type conformation by a functionalizable intrastrand C₂ bridge. Results of CD-based denaturation studies were correlated with calculated (DFT) conformational preferences of the ProM units, revealing that the relative helix stability is mainly governed by an interplay of main-chain preorganization, ring-flip preference, adaptability, and steric effects. Triple helix integrity was proven by crystal structure analysis and binding to HSP47.     

Triple‐Helix‐Stabilizing Effects in Collagen Model Peptides Containing PPII‐Helix‐Preorganized Diproline Modules

Collagen model peptides (CMPs) serve as tools for understanding stability and function of the collagen triple helix and have a potential for biomedical applications. In the past, interstrand cross‐linking or conformational preconditioning of proline units through stereoelectronic effects have been utilized in the design of stabilized CMPs. To further study the effects determining collagen triple helix stability we investigated a series of CMPs containing synthetic diproline‐mimicking modules (ProMs), which were preorganized in a PPII‐helix‐type conformation by a functionalizable intrastrand C₂ bridge. Results of CD‐based denaturation studies were correlated with calculated (DFT) conformational preferences of the ProM units, revealing that the relative helix stability is mainly governed by an interplay of main‐chain preorganization, ring‐flip preference, adaptability, and steric effects. Triple helix integrity was proven by crystal structure analysis and binding to HSP47.     

Role of length-dependent stability of collagen-like peptides

Understanding the structure, folding, and stability of collagen is complex because of its length and variations in the amino acid (AA) sequence composition. It is well known that the basic constituent of the collagen helix is the triplet repeating sequence of the form Gly-X(AA)-Y(AA). On the basis of previous models and with the frequency of occurrence of the triplets, the ((Gly-Pro-Hyp)n)3 (where n is the number of triplets) sequence replicate has been chosen as the model for the most stable form of the collagen-like sequence. With a view to understand the role of sequence length (or the number of triplets) on the stability of collagen, molecular dynamics simulations have been carried out by varying the number of triplet units on the model collagen-like peptides. The results reveal that five triplets are required to form the stable triple helix. Further analysis shows that the intermolecular structural rigidity of the imino acid residues, hydrogen bonding, and water structure around the three chains of the triple helix play the dominant roles on its structure, folding, and stabilization.     

Controlling the morphology of metal-promoted higher ordered assemblies of collagen peptides with varied core lengths

Self-assembling peptides have become an important subclass of next-generation biomaterials. In particular, materials that mimic the properties of collagen have received considerable attention due to the unique properties of natural collagen. Previous peptide-based designs have been successful in generating structures with morphological properties that were primarily determined by the type of self-assembling mechanism. Herein we demonstrate the metal ion-promoted, supramolecular assembly of collagen-based peptide triple helices into distinct morphologies that are controlled by defining the number of Pro-Hyp-Gly repeating units. We synthesized and characterized collagen-based peptides that incorporated either 5, 7, 9, or 11 Pro-Hyp-Gly repeating units. We found that the number of repeating units, and the resulting stability of the collagen triple helix, is intimately linked with the types of assemblies formed. For instance, collagen peptides that did not form a stable triple helix, such as NCoH5, did not participate in supramolecular assembly with added metal ions. Collagen peptides that formed stable triple helices, such as NCoH11, resulted in microsaddle structures with metal-promoted assembly, whereas a highly cross-linked, three-dimensional mesh formed with NCoH7, albeit at a higher metal ion concentration. These data provide evidence that triple helix formation is required for efficient metal-triggered assembly to the observed microstructures.     

Rational design of single-composition ABC collagen heterotrimers

Design of heterotrimeric ABC collagen triple helices is challenging due to the large number of competing species that may be formed. Given the required one amino acid stagger between adjacent peptide strands in this fold, a ternary mixture of peptides can form as many as 27 triple helices with unique composition or register. Previously we have demonstrated that electrostatic interactions can be used to bias the helix population toward a desired target. However, homotrimeric assemblies have always remained the most thermally stable species in solution and therefore comprised a significant component of the peptide mixture. In this work we incorporate complementary modifications to this triple-helical design strategy to destabilize an undesirable competing state while compensating for this destabilization in the desired ABC composition. The result of these modifications is a new ABC triple-helical system with high thermal stability and control over composition, as observed by NMR. An additional set of modifications, which exchanges aspartate for glutamate, results in an overall lowering of stability of the ABC triple helix yet shows further improvement in the system's specificity. This rationally designed system helps to elucidate the rules governing the self-assembly of synthetic collagen triple helices and sheds light on the biological mechanisms of collagen assembly.     

Fluorescence characterization of the thermal stability of collagen mimic peptides

The thermal stability of triple helical structure plays a critical role in collagen biosynthesis, function and degradation. CD technique was utilized to characterize the thermal stability of synthetic collagen mimic peptides. Fluorescence spectroscopy is widely used with easy access all around the world because of its inexpensive instrumentation, low operation cost, easy operation, and high sensitivity. Here we have developed an alternative fluorescence method to detect the thermal stability of collagen mimic peptides. We have demonstrated that fluorescence spectroscopy could measure the thermal stability of collagen mimic peptides with low concentrations under different circumstances. This highly sensitive fluorescence self-quenching assay will greatly expedite the studies of sequence-dependent properties of collagen mimic peptides, and it has great potential in the application of determining the thermal stability of triple helix systems such as collagens, collectins, adiponectin, macrophage scavenger and C1q.     

Insights on the conformational stability of collagen

This review describes work on the conformational stability of the collagen triple helix. In 1994, the structure of collagen was determined at high resolution. Since then, much work has been done on synthetic mimics of collagen that contain host-guest peptides, tethers, peptoid residues, or analogs of the prevalent 4(R)-hydroxy-L-proline residues. This work has revealed much about the chemical basis for collagen stability, and could spawn useful new biomaterials. The literature from 1994 to mid 2001 is reviewed, and 116 references are cited.     

Selective covalent capture of collagen triple helices with a minimal protecting group strategy

Collagens and their most characteristic structural unit, the triple helix, play many critical roles in living systems which drive interest in preparing mimics of them. However, application of collagen mimetic helices is limited by poor thermal stability, slow rates of folding and poor equilibrium between monomer and trimer. Covalent capture of the self-assembled triple helix can solve these problems while preserving the native three-dimensional structure critical for biological function. Covalent capture takes advantage of strategically placed lysine and glutamate (or aspartate) residues which form stabilizing charge–pair interactions in the supramolecular helix and can subsequently be converted to isopeptide amide bonds under folded, aqueous conditions. While covalent capture is powerful, charge paired residues are frequently found in natural sequences which must be preserved to maintain biological function. Here we describe a minimal protecting group strategy to allow selective covalent capture of specific charge paired residues which leaves other charged residues unaltered. We investigate a series of side chain protecting groups for lysine and glutamate in model peptides for their ability to be deprotected easily and in high yield while maintaining (1) the solubility of the peptides in water, (2) the self-assembly and stability of the triple helix, and (3) the ability to covalently capture unprotected charge pairs. Optimized conditions are then illustrated in peptides derived from Pulmonary Surfactant protein A (SP-A). These covalently captured SP-A triple helices are found to have dramatically improved rates of folding and thermal stability while maintaining unmodified lysine–glutamate pairs in addition to other unmodified chemical functionality. The approach we illustrate allows for the covalent capture of collagen-like triple helices with virtually any sequence, composition or register. This dramatically broadens the utility of the covalent capture approach to the stabilization of biomimetic triple helices and thus also improves the utility of biomimetic collagens generally.

A minimal protecting group strategy is developed to allow selective covalent capture of collagen-like triple helices. This allows stabilization of this critical fold while preserving charge–pair interactions critical for biological applications.     

Rules for the design of aza-glycine stabilized triple-helical collagen peptides

The stability of the triple-helical structure of collagen is modulated by a delicate balance of effects including polypeptide backbone geometry, a buried hydrogen bond network, dispersive interfacial interactions, and subtle stereoelectronic effects. Although the different amino acid propensities for the Xaa and Yaa positions of collagen''s repeating (Glycine–Xaa–Yaa) primary structure have been described, our understanding of the impact of incorporating aza-glycine (azGly) residues adjacent to varied Xaa and Yaa position residues has been limited to specific sequences. Here, we detail the impact of variation in the Xaa position adjacent to an azGly residue and compare these results to our study on the impact of the Yaa position. For the first time, we present a set of design rules for azGly-stabilized triple-helical collagen peptides, accounting for all canonical amino acids in the Xaa and Yaa positions adjacent to an azGly residue, and extend these rules using multiple azGly residues. To gain atomic level insight into these new rules we present two high-resolution crystal structures of collagen triple helices, with the first peptoid-containing collagen peptide structure. In conjunction with biophysical and computational data, we highlight the critical importance of preserving the triple helix geometry and protecting the hydrogen bonding network proximal to the azGly residue from solvent. Our results provide a set of design guidelines for azGly-stabilized triple-helical collagen peptides and fundamental insight into collagen structure and stability.

Guidelines for incorporating aza-glycine residues in collagen peptides are presented, detailing their effects on triple-helical thermal stability.     

Metal-assisted stabilization and probing of collagenous triple helices

Collagen model peptides that contain 2,2'-bipyridyl (bpy) ligands were designed and synthesized. The thermal stability of the collagenous triple helix was increased by forming an Fe(II)(bpy-peptide)(3) complex. The chirality of the metal center was shifted to form right-handed Delta-isomers induced by the supercoiling of the peptide moiety. Moreover, the refolding rate of the triple helix was increased in the presence of Fe(II). This metal-coordinating system possesses potential to be used to stabilize the triple-helical conformation as well as to probe the folding status of collagen model peptides.     

Photocontrol of the collagen triple helix: synthesis and conformational characterization of bis-cysteinyl collagenous peptides with an azobenzene clamp

For the photomodulation of the collagen triple helix with an azobenzene clamp, we investigated various collagenous peptides consisting of ideal (Gly-Pro-Hyp) repeats and containing cysteine residues in various positions for a side chain-to-side chain crosslink with a suitable chromophore derivative. Comparative conformational analysis of these cysteine peptides indicated an undecarepeat peptide with two cysteine residues located in the central portion in i and i+7 positions and flanked by (Gly-Pro-Hyp) repeat sequences as the most promising for the cross-bridging experiments. In aqueous alcoholic solution the azobenzene-undecarepeat peptide formed a stable triple helix in equilibrium with the monomeric species as a trans-azobenzene isomer, whereas photoisomerization to the cis isomer leads to unfolding of at least part of the triple helix. Furthermore, the residual supercoiled structure acts like an intermolecular knot, thus making refolding upon cis-to-trans isomerization a concentration-independent fast event. Consequently, these photoswitchable collagenous systems should be well suited for time-resolved studies of folding/unfolding of the collagen triple helix under variable thermodynamic equilibria.