TREN (Tris(2-aminoethyl)amine): an effective scaffold for the assembly of triple helical collagen mimetic structures

A new scaffold, TREN-(suc-OH)(3) where TREN is tris(2-aminoethyl)amine and suc is the succinic acid spacers, was incorporated to assemble triple helices composed of Gly-Nleu-Pro sequences (Nleu denotes N-isobutylglycine). Extensive biophysical studies which include denaturation studies, CD and NMR spectroscopy, and molecular modeling demonstrated that TREN-[suc-(Gly-Nleu-Pro)(n)-NH(2)](3) (n = 5 and 6) form stable triple helical structures in solution. A comparative analysis of TREN-assembled and KTA-assembled collagen mimetics (KTA denotes Kemp triacid, 1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid) indicates that the flexibility of the TREN scaffold is superior to the KTA scaffold in inducing triple helicity. This effect most likely arises from the flexibility of the TREN scaffold which allows the three peptide chains to adjust their register for a tighter triple helical packing.     

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.     

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.     

Synthesis and biological activity of [MeTyr1,MeArg7,D-Leu8]-dynorphin A(1-9)-NHEt and [D-Cys2-Cys5,MeArg7,D-Leu8]-dynorphin A(1-9)-NH2

The opioid activities of [MeTyr1]-Dyn(1-7)-NH2, [MeTyr1,D-Leu8]-Dyn(1-8)-NH2, [MeTyr1,D-Leu8]-Dyn(1-9)-NH2, [MeTyr1,D-Leu8]-Dyn(1-10)-NH2, [MeTyr1,D-Leu8]-Dyn(1-11)-NH2, and [MeTyr1,D-Leu8,12]-Dyn(1-13)-NH2 were examined in the bioassays (guinea pig ileum, mouse vas deferens and rabbit vas deferens). Because [MeTyr1,D-Leu8]-Dyn(1-9)-NH2 showed the most potent opioid activity of the peptides tested, the biological activities of two kinds of Dyn(1-9) analogues, [MeTyr1,MeArg7,D-Leu8]-Dyn(1-9)-NHEt and [D-Cys2-Cys5,MeArg7,D-Leu8]-Dyn(1-9)-NH2 were determined and compared with those of [MeTyr1,MeArg7,D-Leu8]-Dyn(1-8)-NHEt and [D-Cys2-Cys5,MeArg7,D-Leu8]-Dyn(1-8)-NHEt in the three bioassays, in the receptor binding assays, and in the mouse tail pinch test after subcutaneous administration. The results suggest that the extension of the C-terminal in the peptide chain of [MeArg7,D-Leu8]-Dyn(1-8)-NH2 analogues by Arg is ineffective for increasing the kappa-opioid activities, kappa-receptor selectivity and/or analgesic effects of the peptides.     

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!     

Comparison of two thioxopeptide bond photoswitches in insect kinin

<正>The photoisomerization abilities of secondary thioxopeptide bond(CS-NH) and thioxo prolyl bond(CS-N) incorporated into the C-terminal pentapeptide of insect kinin were compared.H-Phe-Phe-Ψ[CS-NH]-D-Ala-Trp-Gly-NH_2 and H-Phe-Tyr-Ψ[CS-N]- Pro-Trp-Gly-NH_2 were studied by UV-vis absorption.The isomerization energy barriers of the two segments,Ac-Phe-Ψ[CS-NH]- D-Ala-NH_2 and Ac-Tyr-Ψ[CS-N]-Pro-NH_2 picked from the two peptides,were calculated using ab initio method.The cis isomer of CS-N is more stable than that of CS-NH due to higher energy barrier,so the former is more suitable in peptide structure-activity relationship studies.     

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.     

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.     

Facile modification of collagen directed by collagen mimetic peptides

Recent widespread interest in the development of engineered tissue and organ replacement therapies has prompted demand for new approaches to immobilize exogenous components to natural collagen. Chemical coupling of synthetic moieties to amino acid side chains has been commonly practiced for such purposes; however, such coupling reactions are difficult to control on large proteins and are generally not conducive to modifying integrated collagen scaffolds that contain live cells and tissues. As an alternative to the conventional "covalent" modification method, we have developed a novel "physical" modification technique that is based on collagen's native ability to associate into a triple-helical molecular architecture. Here, we present a finding that collagen mimetic peptides (CMPs) of sequence -(Pro-Hyp-Gly)x- exhibit strong affinity to both native and gelatinized type I collagen under controlled thermal conditions. We also show that the cell adhesion characteristics of collagen can be readily altered by applying a poly(ethylene glycol)-CMP conjugate to a prefabricated collagen film.     

Approaches to the solid phase of a cyclotriveratrylene scaffold-based tripodal library as potential artificial receptors

Two versatile tripodal cyclotriveratrylene (CTV)-based scaffolds (7 and 9) have been prepared for the solid phase construction of libraries of tripodal artificial synthetic receptors. A 2197-member library of CTV-based tripodal receptor molecules, 20[1-13,1-13,1-13], was prepared on the solid phase using split-mix synthesis. The CTV-based receptors contain three peptide arms; one of them is attached to the solid phase and is different from the other two identical peptide arms.     

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.     

Characterization of conformational adsorbate changes on a tissue-derived substrate using Fourier transform infrared spectroscopy

Fourier transform infrared (FT-IR) spectroscopy is utilized to observe adsorbate interactions with a tissue-derived collagen scaffold extracted from the Bruch's membrane of pig eyes. The characterization includes conformational changes in isoleucine, polyisoleucine, collagen-binding peptide, RGD-tagged collagen-binding peptide, and laminin after adsorption onto the substrate. Isotopically labeled isoleucine is further utilized to understand changes in the biomolecular structure upon binding to a tissue-derived surface. The adsorbates associated with the collagen scaffold predominately through hydrophobic interactions and hydrogen bonding. The results of this study can be used to improve our understanding of surface chemistry changes during the engineering of biomimetic scaffolds before and after biomolecule adsorption.     

Tricine as a convenient scaffold for the synthesis of C-terminally branched collagen-model peptides

A novel and convenient method for the synthesis of C-terminally branched collagen-model peptides has been achieved using tricine (N-[tris(hydroxymethyl)methyl]glycine) as a branching scaffold and 1,2-diaminoethane or 1,4-diaminobutane as a linker. The peptide sequence was incorporated directly onto the linker and scaffold during solid-phase synthesis without additional manipulations. The resulting branched triple-helical peptides exhibited comparable thermal stabilities to the parent, unbranched sequence, and served as substrates for matrix metalloproteinase-1 (MMP-1). The tricine-based branch reported herein represents the simplest synthetic scaffold for the convenient synthesis of covalently linked homomeric collagen-model triple-helical peptides.     

Metal clips induce folding of a short unstructured peptide into an alpha-helix via turn conformations in water. Kinetic versus thermodynamic products

Short peptides corresponding to two to four alpha-helical turns of proteins are not thermodynamically stable helices in water. Unstructured octapeptide Ac-His1-Ala2-Ala3-His4-His5-Glu6-Leu7-His8-NH(2) (1) reacts with two [Pd((15)NH(2)(CH(2))(2)(15)NH(2))(NO(3))(2)] in water to form a kinetically stable intermediate, [[Pden](2)[(1,4)(5,8)-peptide]](2), in which two 19-membered metallocyclic rings stabilize two peptide turns. Slow subsequent folding to a thermodynamically more stable two-turn alpha-helix drives the equilibrium to [[Pden](2)[(1,5)(4,8)-peptide]] (3), featuring two 22-membered rings. This transformation from unstructured peptide via turns to an alpha-helix suggests that metal clips might be useful probes for investigating peptide folding.     

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.     

Dual-peptide-modified alginate hydrogels for the promotion of angiogenesis

The ability to supply suitable blood vessel system is a major challenge for artificial thick tissue engineering. Angiogenesis is a key point during the process of microvascular formation. Many bioactive molecules such as extra cellular matrix(ECM) proteins and adhesion peptides derived from the ECM are applied to promote angiogenesis. In this work, two adhesion peptides, YIGSR and REDV, were selected to modify sodium alginate(ALG) to obtain YIGSR- and REDV-alginate conjugates(ALG-YIGSR, and ALG-REDV, respectively). We mixed the two peptide-conjugates together in a series of concentration ratios to prepare bioactive surfaces for in vitro studies and hydrogel scaffolds for in vivo studies. In vitro studies showed that surfaces modified with 1.09 pmol/mm2 peptide had the best affinity to human umbilical vein endothelial cells(HUVECs) than that with high or low concentrations of peptides. In addition, surfaces modified with dual peptides could significantly promote HUVECs proliferation, where ALG-YIGSR:ALG-REDV at a mole ratio of 5:1 exhibited the best enhancement ability. Furthermore, the in vivo angiogenesis results demonstrated that hydrogel scaffolds composed of mixed ALG-YIGSR and ALG-REDV at the 5:1 ratio had angiogenic induction potential by stimulating new blood vessel formation, and showed higher blood vessel density than scaffolds composed of a single peptide. These results demonstrated that a mixed combination of peptide alginate conjugates could be a potential scaffold to stimulate and induce angiogenesis in tissue engineering applications.     

Computational study of conformational preferences of thioamide-containing azaglycine peptides

The effect of thioamide substitution on the conformational stability of an azaglycine-containing peptide, For-AzaGly-NH2 (1), was investigated for the sake of finding possible applications by using ab initio and DFT methods. As model compounds, For-[psiCSNH]-AzaGly-NH2 (2), For-AzaGly-[psiCSNH]-NH2 (3), and For-[psiCSNH]-AzaGly-[psiCSNH]-NH2 (4) were used. Two-dimensional phi-psi potential energy surfaces (PESs) for 2-4 were calculated at the B3LYP/6-31G*//HF/6-31G* level in gas (epsilon = 1.0) and in water (epsilon = 78.4) by applying the isodensity polarizable continuum model (IPCM) method. On the basis of these PESs, the minimum energy conformations for 2-4 were characterized at the B3LYP level with 6-31G*, 6-311G**, and 6-31+G** basis sets. The remarkable structural effect of thioamide substitution for 2-4 is that beta-strand structure is observed as a global or local minimum. The minima of 2-4 are also compared with those for glycine and thioamide-containing glycine peptides. Our theoretical results demonstrate that compounds 2-4 would be used to design controllable secondary structures.     

Entropic control of the relative stability of triple-helical collagen peptide models

Herein, we show that current methodologies in atomistic simulations can yield reliable standard free energy values in aqueous solution for the transition from the dissociated monomeric form to the triple-helix state of collagen model peptides. The calculations are performed on a prototypical highly stable triple-helical peptide, [(Pro-Hyp-Gly)10]3 (POG10), and on the so-called T3-785 triple-helix mimicking a fragment from the type III human collagen, which is more thermally labile. On the basis of extensive MD simulations in explicit solvent followed by molecular-mechanical and electrostatic Poisson-Boltzmann calculations complemented with an accurate estimation of the nonpolar contributions to solvation, the computed free energy change for the aggregation processes of the POG10 and T3-785 peptides leading to their triple-helices is -6.6 and -6.1 kcal/mol, respectively. For POG10, this value is in agreement with differential scanning calorimetric data. However, it is shown that conformational entropy, which is estimated by means of an expansion of mutual information functions, preferentially destabilizes the triple-helical state of T3-785 by around 4.6 kcal/mol, thus explaining its lower thermal stability. Altogether, our computational results allow us to ascertain, for the first time, the actual thermodynamic forces controlling the absolute and relative stability 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.     

Single turn peptide alpha helices with exceptional stability in water

Cyclic pentapeptides are not known to exist in alpha-helical conformations. CD and NMR spectra show that specific 20-membered cyclic pentapeptides, Ac-(cyclo-1,5) [KxxxD]-NH(2) and Ac-(cyclo-2,6)-R[KxxxD]-NH(2), are highly alpha-helical structures in water and independent of concentration, TFE, denaturants, and proteases. These are the smallest alpha-helical peptides in water.