Synthetic collagen heterotrimers: structural mimics of wild-type and mutant collagen type I

Collagen type I is an AAB heterotrimer assembled from two alpha1 chains and one alpha2 chain. Missense mutations in either of these chains that substitute a glycine residue in the ubiquitous X-Y-Gly repeat with a bulky amino acid leads to osteogenesis imperfecta (OI) of varying severity. These mutations have been studied in the past using collagen-like peptide homotrimers as a model system. However, homotrimers, which by definition will contain glycine mutations in all the three chains, do not accurately mimic the mutations in their native form and result in an exaggerated effect on stability and folding. In this article, we report the design of a novel model system based upon collagen-like heterotrimers that can mimic the glycine mutations present in either the alpha1 or alpha2 chains of type I collagen. This design utilizes an electrostatic recognition motif in three chains that can force the interaction of any three peptides, including AAA (all same), AAB (two same and one different), or ABC (all different) triple helices. Therefore, the component peptides can be designed in such a way that glycine mutations are present in zero, one, two, or all three chains of the triple helix. With this design, we for the first time report collagen mutants containing one or two glycine substitutions with structures relevant to native forms of OI. Furthermore, we demonstrate the difference in thermal stability and refolding half-life times between triple helices that vary only in the frequency of glycine mutations at a particular position.     

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.     

Self-assembled heterotrimeric collagen triple helices directed through electrostatic interactions

Collagen, a fibrous protein, is an essential structural component of all connective tissues such as cartilage, bones, ligaments, and skin. Type I collagen, the most abundant form, is a heterotrimer assembled from two identical alpha1 chains and one alpha2 chain. However, most synthetic systems have addressed homotrimeric triple helices. In this paper we examine the stability of several heterotrimeric collagen-like triple helices with an emphasis on electrostatic interactions between peptides. We synthesize seven 30 amino acid peptides with net charges ranging from -10 to +10. These peptides were mixed, and their ability to form heterotrimers was assessed. We successfully show the assembly of five different AAB heterotrimers and one ABC heterotrimer. The results from this study indicate that intermolecular electrostatic interactions can be utilized to direct heterotrimer formation. Furthermore, amino acids with poor stability in collagen triple helices can be "rescued" in heterotrimers containing amino acids with known high triple helical stability. This mechanism allows collagen triple helices to have greater chemical diversity than would otherwise be allowed.     

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.     

Computational design of a collagen A:B:C-type heterotrimer

We have successfully designed an A:B:C collagen peptide heterotrimer using an automated computational approach. The algorithm maximizes the energy gap between the target and competing misfolded states while enforcing a minimum target stability. Circular dichroism (CD) measurements confirm that all three peptides are required to form a stable, structured triple helix. This study highlights the power of automated computational design, providing model systems to probe the biophysics of collagen assembly and developing general methods for the design of fibrous proteins.     

NMR monitoring of chain-specific stability in heterotrimeric collagen peptides

NMR spectroscopy is used to investigate the heterotrimeric nature of a collagen model peptide. Two distinct peptide chains (A and B) were synthesized to model a site in heterotrimeric basement membrane type IV collagen. For NMR studies, four amino acids in the B chain were labeled with 15N/13C. Circular dichroism spectroscopy and differential scanning calorimetry thermal stability results on a solution with both A and B peptides (molar ratio 2A:1B) are consistent with the presence of one heterotrimeric triple-helical molecular species. Heteronuclear single quantum coherence experiments on homotrimers of the B peptide show trimer peaks which disappear at temperatures higher than 10 degrees C, while the 2A:1B mixture has trimer peaks with increased stability and altered chemical shifts. The reduction in the number of Leu trimer peaks from three to one and the increased stability of trimer resonances confirm the participation of B chains in an AAB heterotrimer molecule.     

Metal-assisted assembly and stabilization of collagen-like triple helices

Single-chain and TRIS-assembled collagen mimetic peptide structures incorporating catechol groups were synthesized. When 1/3 equiv of Fe3+ was added to the single-chain compound in 50 mM CAPS buffer (pH 10), the 1:3 Fe3+-catechol complex that formed acted as an N-terminal scaffold to assemble the triple helix. When 1 equiv of Fe3+ was added to the TRIS-assembled compound in the buffer solution, the Fe3+-catechol complex acted as an extra C-terminal scaffold, which lead to a triple helix with both termini tethered. The formation of this C-terminal complex increased the Tm by a remarkable 22 degrees C!     

The role of cystine knots in collagen folding and stability,part I. Conformational properties of (Pro-Hyp-Gly)5 and (Pro-(4S)-FPro-Gly)5 model trimers with an artificial cystine knot

In analogy to the cystine knots present in natural collagens, a simplified disulfide cross-link was used to analyse the conformational effects of a C-terminal artificial cystine knot on the folding of collagenous peptides consisting of solely (Pro-Hyp-Gly) repeating units. Assembly of the alpha chains into a heterotrimer by previously applied regioselective disulfide-bridging strategies failed because of the high tendency of (Pro-Hyp-Gly)(5) peptides to self-associate and form homotrimers. Only when side-chain-protected peptides were used, for example in the Hyp(tBu) form, and a new protection scheme was adopted, selective interchain-disulfide cross-linking into the heterotrimer in organic solvents was successful. This unexpected strong effect of the conformational properties on the efficiency of well-established reactions was further supported by replacing the Hyp residues with (4S)-fluoroproline, which is known to destabilise triple-helical structures. With the related [Pro-(4S)-FPro-Gly](5) peptides, assembly of the heterotrimer in aqueous solution proceeded in a satisfactory manner. Both the intermediates and the final fluorinated heterotrimer are fully unfolded in aqueous solution even at 4 degrees C. Conversely, the disulfide-crossbridged (Pro-Hyp-Gly)(5) heterotrimer forms a very stable triple helix. The observation that thermal unfolding leads to scrambling of the disulfide bridges was unexpected. Although NMR experiments support an extension of the triple helix into the cystine knot, thermolysis is not associated with the unfolding process. In fact, the unstructured fluorinated trimer undergoes an equally facile thermodegradation associated with the intrinsic tendency of unsymmetrical disulfides to disproportionate into symmetrical disulfides under favourable conditions. The experimental results obtained with the model peptides fully support the role of triple-helix nucleation and stabilisation by the artificial cystine knot as previously suggested for the natural cystine knots in collagens.     

The new science of protein mimetics

New chemistries have been developed for de novo protein design. Protein mimetics of different structural and functional properties such as synthetic peptide ligases and Dn symmetrical helical bundles have been reported. The Template-Assembled Synthetic Protein (TASP) method (as well as the ßMolecular Kit' approach) has also been utilized to prepare protein-like molecules. Here we report the synthesis of single chain, scaffold (TRIS)- and dendrimer-assembled collagen mimetics composed of the Gly-Nleu-Pro sequence where Nleu denotes N-isobutyl glycine. From the CD spectra and the thermal denaturation studies it can be seen that the collagen mimetics prepared form stable triple helices except the single chain structure. Furthermore, the 162-residue collagen mimetic dendrimer exhibits enhanced triple helical stability compared to the equivalent scaffold-terminated structure by an increase in the melting temperature in both H₂O and 2:1 ethylene glycol/H₂O (4°C and 12°C respectively). The concentration dependence for the melting transition of the collagen mimetic dendrimer was measured from which it was determined that the stabilization effect arises from the intramolecular clustering of the triple helical arrays about the core structure. This ensemble excludes solvent from the interior portion of the array which stabilizes the triple helix cluster.     

Peptide tic-tac-toe: heterotrimeric coiled-coil specificity from steric matching of multiple hydrophobic side chains

Specific coiled-coil heterotrimers result from steric matching of hydrophobic core side chains. A 2:1 heterotrimer is formed by peptides containing alanine or cyclohexylalanine, respectively, at a central core residue. Detailed thermodynamic analysis reveals that the designed complex is considerably more stable than the corresponding alanine homotrimer (deltaT(m) = 25 degrees C, deltadeltaG(unf) = 4.5 kcal/mol), while control complexes with naphthylalanine or cyclopropylalanine peptides are much less stable. However, the cyclohexylalanine homotrimer is of comparable stability to the 2:1 complex, prompting an investigation of multiply substituted peptides. A specific 1:1:1 heterotrimer is formed from three independent peptide strands, each bearing one large (cyclohexylalanine) and two small (alanine) side chains at the same three core positions but in different order. The combined impact of three substitutions improves specificity to the point where each pure peptide and all pairwise equimolar mixtures form significantly less stable complexes (deltaTm = 22-24 degrees C). The capacity for specific complex formation governed by multiple unnatural core side chains should facilitate design of numerous new peptide assemblies.     

The role of cystine knots in collagen folding and stability,part II. Conformational properties of (Pro-Hyp-Gly)n model trimers with N- and C-terminal collagen type III cystine knots

In mature collagen type III the homotrimer is C-terminally cross-linked by an interchain cystine knot consisting of three disulfide bridges of unknown connectivity. This cystine knot with two adjacent cysteine residues on each of the three alpha chains has recently been used for the synthesis and expression of model homotrimers. To investigate the origin of correct interchain cysteine pairings, (Pro-Hyp-Gly)(n) peptides of increasing triplet number and containing the biscysteinyl sequence C- and N-terminally were synthesised. The possibilities were that this origin may be thermodynamically coupled to the formation of the collagen triple helix as happens in the oxidative folding of proteins, or it could represent a post-folding event. Only with five triplets, which is known to represent the minimum number for self-association of collagenous peptides into a triple helix, air-oxidation produces the homotrimer in good yields (70 %), the rest being intrachain oxidised monomers. Increasing the number of triplets has no effect on yield suggesting the formation of kinetically trapped intermediates, which are not reshuffled by the glutathione redox buffer. N-terminal incorporation of the cystine knot is significantly less efficient in the homotrimerisation step and also in terms of triple-helix stabilisation. Compared to an artificial C-terminal cystine knot consisting of two interchain disulfide bridges, the collagen type III cystine knot produces collagenous homotrimers of remarkably high thermostability, although the concentration-independent refolding rates are not affected by the type of disulfide bridging. Since the natural cystine knot allows ready access to homotrimeric collagenous peptides of significantly enhanced triple-helix thermostability it may well represent a promising approach for the preparation of collagen-like innovative biomaterials. Conversely, the more laborious regioselectively formed artificial cystine knot still represents the only synthetic strategy for heterotrimeric collagenous peptides.     

Fe(III)-binding collagen mimetics

The synthesis and characterization of hydroxamic acid containing single-chain and TRIS-assembled (where TRIS is tris(carboxyethoxymethyl)aminomethane) collagen mimetics are reported. We have engineered an Fe(III)-binding domain by placing a hydroxamic acid group at the C termini of collagen mimetic chains composed of the Gly-Pro-NLeu sequence. The circular dichroism spectra and thermal denaturation studies show an enhancement in triple-helical thermal stability upon the addition of Fe(III) for the TRIS-assembled structure. No triple-helical structure was detected for the single-chain collagen mimetic. From the absorbance shown in the UV-vis spectra, we believe that the thermal stabilization of the triple helix is the direct result of a coordination complex between Fe(III) and the hydroxamate groups tethered to the C termini of the collagen mimetic peptide chains.     

Specific control of peptide assembly with combined hydrophilic and hydrophobic interfaces

Designed coiled-coil heterotrimers are described whose assembly is governed by both hydrophobic and hydrophilic forces. Sterically matched hydrophobic core side-chain packing of alanine and cyclohexylalanine has been shown to promote formation of a 1:1:1 heterotrimer. Manipulation of hydrophilic glutamic acid (Glu)/lysine (Lys) pairs at each of three helical contact interfaces provides a secondary recognition mechanism. Peptides with matched cores and hydrophilic contacts form stable heterotrimers (DeltaG(unf) at 25 degrees C = 17.93 kcal/mol; MW(app) = 11362 vs 11563 calcd for trimer), as do those with a single Lys/Lys (but not Glu/Glu) interface. The additional specificity engendered by simultaneous operation of two interfaces was used to design a system in which six different peptides are mixed to form three specific and independent heterotrimers in the same solution.     

Selective formation of AAB- and ABC-type heterotrimeric alpha-helical coiled coils

The alpha-helical coiled coils have a representative amino acid sequence of (abcdefg)(n) heptad repeats. We previously reported that two peptides named IZ-2A and IZ-2W formed an (IZ-2A)(2)/IZ-2W heterotrimer with an Ala-Ala-Trp interaction in the hydrophobic core. In this paper, we describe the selective formation of AAB- and ABC-type heterotrimers. To increase the selectivity of the AAB-type heterotrimeric formation, Lys residues at the f position were mutated to either an Ala or a Gln residue to form IZ-2A(fA) or IZ-2W(fQ). Separately, both IZ-2A(fA) and IZ-2W(fQ) have a random structure at pH 7 and 20 degrees C. However, together IZ-2A(fA) and IZ-2W(fQ) form a 2:1 complex with a thermal transition midpoint (Tm) of 48 degrees C. This procedure was applied to prepare the ABC-type heterotrimer, in which two sets of Ala-Ala-Trp interactions were designed in the hydrophobic core. Interhelical interaction between the e and g positions and the alpha-helical propensity of the amino acid at the f position were also considered in the design. The resultant three peptides selectively formed the ABC-type heterotrimer with a Tm of 51 degrees C. Other peptide combinations had random coil properties.     

Peptides that anneal to natural collagen in vitro and ex vivo

Collagen comprises ? of the protein in humans and ? of the dry weight of human skin. Here, we implement recent discoveries about the structure and stability of the collagen triple helix to design new chemical modalities that anchor to natural collagen. The key components are collagen mimetic peptides (CMPs) that are incapable of self-assembly into homotrimeric triple helices, but are able to anneal spontaneously to natural collagen. We show that such CMPs containing 4-fluoroproline residues, in particular, bind tightly to mammalian collagen in vitro and to a mouse wound ex vivo. These synthetic peptides, coupled to dyes or growth factors, could herald a new era in assessing or treating wounds.     

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.     

Peptide bond isosteres: ester or (E)-alkene in the backbone of the collagen triple helix

[structure: see text] Collagen is the most abundant protein in animals. Interstrand N-H...O=C hydrogen bonds between backbone amide groups form a ladder in the middle of the collagen triple helix. Isosteric replacement of the hydrogen-bond-donating amide with an ester or (E)-alkene markedly decreases the conformational stability of the triple helix. Thus, this recurring hydrogen bond is critical to the structural integrity of collagen. In this context, an ester isostere confers more stability than does an (E)-alkene.     

Structurally Homogeneous Nanosheets from Self‐Assembly of a Collagen‐Mimetic Peptide

A collagen‐mimetic peptide, NSIII, has been designed with three sequential blocks having positive, neutral, and negative charges, respectively. The non‐canonical imino acid, (2S,4S)‐4‐aminoproline (amp), was used to specify the positive charges at the Xaa positions of (Xaa‐Yaa‐Gly) triads in the N‐terminal domain of NSIII. Peptide NSIII underwent self‐assembly from aqueous solution to form a highly homogeneous population of nanosheets. Two‐dimensional crystalline sheets formed in which the length of the peptide defined the height of the sheets. These results contrasted with prior results on a similar multi‐domain collagen‐mimetic polypeptides in which the sheets had highly polydisperse distribution of sizes in the (x/y)‐ and (z)‐dimensions. The structural differences between the two nanosheet assemblies were interpreted in terms of the relative stereoelectronic effects of the different aminoproline derivatives on the local triple helical conformation of the peptides.     

How stable is a collagen triple helix? An ab initio study on various collagen and β‐sheet forming sequences

Collagen forms the well characterized triple helical secondary structure, stabilized by interchain H-bonds. Here we have investigated the stability of fully optimized collagen triple helices and beta-pleated sheets by using first principles (ab initio and DFT) calculations so as to determine the secondary structure preference depending on the amino acid composition. Models composed of a total of 18 amino acid residues were studied at six different amino acid compositions: (i) L-alanine only, (ii) glycine only, (iii) L-alanines and glycine, (iv) L-alanines and D-alanine, (v) L-prolines with glycine, (vi) L-proline, L-hydroxyproline, and glycine. The last two, v and vi, were designed to mimic the core part of collagen. Furthermore, ii, iii, and iv model the binding and/or recognition sites of collagen. Finally, i models the G-->A replacement, rare in collagen. All calculated structures show great resemblance to those determined by X-ray crystallography. Calculated triple helix formation affinities correlate well with experimentally determined stabilities derived from melting point (T(m)) data of different collagen models. The stabilization energy of a collagen triple helical structure over that of a beta-pleated sheet is 2.1 kcal mol(-1) per triplet for the [(-Pro-Hyp-Gly-)(2)](3) collagen peptide. This changes to 4.8 kcal mol(-1) per triplet of destabilization energy for the [(-Ala-Ala-Gly-)(2)](3) sequence, known to be disfavored in collagen. The present study proves that by using first principles methods for calculating stabilities of supramolecular complexes, such as collagen and beta-pleated sheets, one can obtain stability data in full agreement with experimental observations, which envisage the applicability of QM in molecular design.     

pH-triggered strand exchange in coiled-coil heterotrimers

The capacity for pH-triggered strand exchange in designed coiled-coil heterotrimers is demonstrated. Systems employing both hydrophobic core (steric matching) and hydrophilic interface (electrostatic matching) design principles assemble into specific 1:1:1 heterotrimers. Alteration of pH creates electrostatic mismatches, inducing strand exchange in the presence of a suitable replacement peptide. Complexes with one Lys/Lys interface, favored at neutral to high pH, can be transformed to ones with a Glu/Glu contact by lowering pH and adding an appropriate new binding partner. The need to simultaneously maintain matched core alignments enforces specificity in this exchange, such that only a single specific peptide is replaced. These principles have subsequently been applied to the design of dynamically triggered cross-linked structures, in which a bifunctional disulfide-tethered peptide can cross-link two heterotrimers. Both formation and disruption of the cross-link are under pH control.