Gelatin hydrogels cross-linked with bis(vinylsulfonyl)methane (BVSM): 1. The chemical networks

This paper deals with chemical gelation of gelatin in the presence of a cross-linker, bis(vinylsulfonyl)methane (BVSM), which is able to create covalent C-N bonds with amine groups. The investigation is performed at 40 degrees C, where no triple helices are present. Gelatin is in random coil conformation. The influence of various parameters (gelatin concentration, cross-linker concentration, and pH (number of reacting sites along the gelatin chain)) was examined. Gel formation was followed by rheological and thermodynamic measurements (microcalorimetry) versus time (kinetic measurements). Furthermore, the storage moduli were compared to the number of links formed in the course of gelation. The experiments show that, within the experimental range investigated, a fully homogeneous network is not reached; the chemical gels, even upon completion of the reactions, are still in the critical domain, near the threshold. A power law behavior was put in evidence for the shear modulus versus the distance to the gel point, expressed as the concentration of links per gelatin chain. The exponent (f = 3.4 +/- 0.3) is close to that expected for the vulcanization of long chains. The storage moduli can be superposed on a single curve where the abscissa is the product of the number of C-N links per unit volume and the gelatin concentration at an exponent equal to -0.76 +/- 0.03. This exponent suggests the role of entanglements for interchain cross-linking.     

Effect of sodium poly(styrene sulfonate) on thermoreversible gelation of gelatin

The effect of an added polyanion, sodium poly(styrene sulfonate) (NaPSS), on the thermoreversible gelation and remelting of gelatin gels has been investigated by polarimetry and rheology. The presence of NaPSS can either enhance or reduce collagenlike helix formation, depending on the polymer concentration relative to that of gelatin and the gelation temperature. At temperatures < 20°C, the helical content is reduced by increasing the amount of added NaPSS, demonstrating the disruption of helical structure of gelatin by the polyanion. Synchronous measurements of optical rotation and modulus at 25°C, in both gelation and remelting, indicate that the optical rotation at the gel point for the pure gelatin is lowered on addition of NaPSS. At low frequency, the storage modulus of gelatin is increased by the addition of a small amount of NaPSS relative to that of gelatin, but decreased with excess NaPSS. The mechanical properties of gelatin with and without NaPSS will be discussed in light of the competition between network junction formation by strands of triple helices among gelatin chains and temporary ionic crosslinking between gelatin and the polyanion. © 1999 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 37: 2287–2295, 1999     

Molecular cable-like 1-D iodic spiral chains covered with triple helices stabilized in guest-included chiral porous framework

The supramolecular crystal {[Pr(DMFA)](3)[Ni(II)(Hbim)(3)](2)I}(n) with intricate chiral networks of [Ni(II)(Hbim)(3)](-) molecules is reported. It includes a cationic architecture as a guest, constructed from chiral nanotubes that penetrate I(-) chains with spiral channels wrapped by triple helices. The I(-) chains have AC conductivity in crystals like a molecular cable.     

Aminoglycoside-nucleic acid interactions: remarkable stabilization of DNA and RNA triple helices by neomycin

The stabilization of poly(dA).2poly(dT) triplex, a 22-base DNA triplex, and poly(rA).2poly(rU) triple helix by neomycin is reported. The melting temperatures, the association and dissociation kinetic parameters, and activation energies (E(on) and E(off)) for the poly(dA).2poly(dT) triplex in the presence of aminoglycosides and other triplex binding ligands were determined by UV thermal analysis. Our results indicate that: (i) neomycin stabilizes DNA triple helices, and the double helical structures composed of poly(dA).poly(dT) are virtually unaffected. (ii) Neomycin is the most active and triplex-selective stabilization agent among all aminoglycosides, previously studied minor groove binders, and polycations. Its selectivity (DeltaT(m3-->2) vs DeltaT(m2)(-->)(1)) exceeds most intercalating drugs that bind to triple helices. (iii) Neomycin selectively stabilizes DeltaT(m3)(-->)(2) for a mixed 22-base DNA triplex containing C and T bases in the pyrimidine strand. (iv) The rate constants of formation of triplex (k(on)) are significantly enhanced upon increasing molar ratios of neomycin, making triplex association rates closer to duplex association rates. (v) E(on) values become more negative upon increasing concentration of aminoglycosides (paromomycin and neomycin). E(off) values do not show any change for most aminoglycosides except neomycin. (vi) Aminoglycosides can effectively stabilize RNA [poly(rA).2poly(rU)] triplex, with neomycin[being one of the most active ligands discovered to date (second only to ellipticine). (vii) The stabilization effect of aminoglycosides on triple helices is parallel to their toxic behavior, suggesting a possible role of intramolecular triple helix (H-DNA) stabilization by the aminoglycosides.     

Interfacial rheological studies of gelatin-sodium dodecyl sulfate complexes adsorbed at the air-water interface

The dilational rheological behavior of gelatin molecules adsorbed at the air-water interface has been studied as a function of sodium dodecyl sulfate (SDS) concentration for a 7 wt % gelatin-SDS solution at 40 degrees C. Binding of SDS molecules to the gelatin strands disrupts the cross-linked network structure of adsorbed gelatin molecules and results in a reduction of the surface elastic modulus of the adsorbed layer that continues until the bulk SDS concentration reaches 1 mM. Beyond this SDS concentration, the dilational rheological properties of the adsorbed gelatin layer are indistinguishable from those of pure SDS adsorbed layers.     

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.     

Thermoreversible gelation strongly coupled to coil-to-helix transition of polymers

This paper theoretically studies thermoreversible gelation driven by aggregation of helices formed on the polymer chains. Two fundamentally different cases of (i) multiple association of single helices and (ii) association by multiple helices with multiplicity k (such as double helices (k=2), triple helices (k=3), etc.) are treated on the basis of different equations. The helix length distribution on a polymer chain (or assemble of chains for multiple helices) is derived as a function of polymer concentration and temperature. Theoretical calculation of the total helix content in the solution is compared with experimental data of optical rotation in iota-carrageenan solutions at different polymer concentrations. It is shown that at low temperature there is a sharp transition from network to bundle state (pair, triplet, etc.). To confirm such a network/pairing transition, we carried out Monte Carlo simulation of polymer solution in which hydrogen-bonded zipper-like cross-links are formed.     

Compactization of rigid-chain amphiphilic macromolecules with local helical structure

Conformational characteristics of amphiphilic macromolecules with secondary local helical structuring are studied by the method of molecular dynamics for different properties of a helix (bending angles between neighboring vectors of the bond and internal rotation angle) and different rigidities of its fixation. Extended helices with high distances between helical turns and dense helices in which neighboring turns directly adjoin each other are studied. As the quality of a solvent deteriorates, extended helices experience a well-pronounced coil-globule transition, whose amplitude increases with an increase in chain rigidity, while the dimensions of dense helices gradually change. In a poor solvent, extended helices formed “collagen-like” structures, flexible chains of dense helices produce hairpin structures, and rigid macromolecules of dense helices form rodlike globules with an almost ideal local helical order. Independently of helix parameters, a deterioration in solvent quality leads to stabilization of the local secondary structure.     

Gelatin hydrogels cross-linked with bisvinyl sulfonemethyl. 2. The physical and chemical networks

This paper deals with the physical and the chemical gelation of gelatin in the presence of a reactant, bisvinyl sulfonemethyl (BVSM). The strategy of this investigation is to separate the contributions of the two types of cross-links in order to deduce the resultant elasticity of the network. In addition, the question raised by several authors concerning an increase of the thermal stability of the triple helices in the presence of cross-links was examined by using several techniques. In this study, the concentration of gelatin and BVSM were kept constant, while the influence of the thermal protocols was put in evidence. The gel formation was followed by rheological, thermodynamic (microcalorimetry), and optical spectroscopy (optical rotation) measurements. The results demonstrate the large differences which arise on the storage moduli by changing the thermal protocols. Cross-linking of the networks in the presence of the triple helices induce a heterogeneous repartition of the bonds, which can form along the triple helices and at the end of the sequences. Consequently, the rubber like network obtained by denaturation of the triple helices is still reminiscent of the initial twist of the chains, and a large modulus is observed, as if rigid segments were still present (storage modulus 10 times larger than for random cross-linking). The hydrogels have an elastic modulus which is larger that the addition of the physical and chemical contributions. The interpretation of the network elasticity is based on the predominant role of the rigid rods of triple helices, where the BVSM cross-links can either modify the ratio between the apparent length and distance between rods, l/d, and/or increase the rigidity of the interchain connections, which are loose coils for the physical gels. The hydrogels investigated have a network which is still close to the percolation threshold of the physical gel, and therefore, the statistical models known for well developed networks cannot be directly validated in these experimental conditions.     

Large deformation rheology of gelatin gels

We studied the linear and the non-linear elastic behaviour, the breaking stress and breaking strain of gelatin gels as a function of a number of experimental conditions: gelatin concentration, gelatin bloom value, ageing time, ageing temperature, pH, NaCl and CaCl₂ concentration, whey protein concentration, the amount of pre-shearing, strain rate or compression speed, using both shear deformation and compression. We analyzed the stress-strain curves using the BST-equation (Blatz et al., Trans. Soc. Rheol. 18, (1974) 145) and extracted a parameter that characterizes the linear elastic behaviour at small deformations (the moduli E or G) and one that characterizes the non-linear elastic behaviour at large deformations (the elasticity parameter n). The phenomenological BST equation describes rheological experiments adequately both in shear deformation and in compression.We found that the modulus correlates with the breaking stress. For the non-linear elastic properties of gelatin, we found that the elasticity parameter n correlates with the breaking strain Qualitatively, the non-linear elastic properties can be explained by assuming that the gelatin chains are partially in a crystalline triple helix state (the cross-links) and partially in a random coil state (the network bonds): the more extensive the rigid cross-link regions, the shorter and more stretched the network bonds become as a result of an externally applied deformation. The network bonds behave as anharmonic springs under extreme extension.Manipulation of the breaking strain was attempted in two ways: (i) by changing the (non-linear) elasticity parameter of the gel: this is possible by using a gel that has been further aged; (ii) by adding defects to the gel structure: this is possible by either pre-shearing the gel or by adding whey protein particles. The pre-shearing gives rise to a temporary effect, the addition of whey protein particles to a permanent effect.     

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.     

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.     

Effects of hydroxy compounds on gel formation of gelatin

 Network formation of gelatin gel is known to consist of three-dimensionally cross-linked triple helices among polypeptide chains. The effects of added low molecular weight mono-ols, diols and polyols on the higher-order structure formation of gelatin chains were investigated using the following measurements: melting temperature, viscoelasticity and spin-lattice relaxation time (T ₁) of H¹⁷ ₂O of gels, and circular dichroism spectra of diluted gelatin solutions. Furthermore, hydration behaviors of these hydroxy compounds were evaluated from the dynamic hydration numbers (n _DHN) derived from T ₁ of H¹⁷ ₂O in the solutions. It was found that network structures of gelatin gels containing hydoxy compounds were influenced by the number and position of hydroxyl groups as well as the number of carbon atoms of these coexisting compounds. The effect of hydroxyl groups of hydroxy compounds was considered to stabilize the helices among gelatin chains. Especially, the addition of polyols with large number of hydroxyl groups increased the number of cross-linking junctions in the gel networks, which consist of the aggregation among the helices. On the contrary, the effect of carbon atoms of hydroxy compounds is to disturb the formation of the helices and the aggregation among the helices. Received: 18 April 1996/Accepted: 23 July 1996     

Stereoelectronic and steric effects in the collagen triple helix: toward a code for strand association

Collagen is the most abundant protein in animals. The protein consists of a helix of three strands, each with sequence X-Y-Gly. Natural collagen is most stable when X is (2S)-proline (Pro) and Y is (2S,4R)-4-hydroxyproline (4R-Hyp). We had shown previously that triple helices in which X is (2S,4S)-4-fluoroproline (4S-Flp) or Y is (2S,4R)-4-fluoroproline (4R-Flp) display hyperstability. This hyperstability arises from stereoelectronic effects that preorganize the main-chain dihedral angles in the conformation found in the triple helix. Here, we report the synthesis of strands containing both 4S-Flp in the X-position and 4R-Flp in the Y-position. We find that these strands do not form a stable triple helix, presumably because of an unfavorable steric interaction between fluoro groups on adjacent strands. Density functional theory calculations indicate that (2S,3S)-3-fluoroproline (3S-Flp), like 4S-Flp, should preorganize the main chain properly for triple-helix formation but without a steric conflict. Synthetic strands containing 3S-Flp in the X-position and 4R-Flp in the Y-position do form a triple helix. This helix is, however, less stable than one with Pro in the X-position, presumably because of an unfavorable inductive effect that diminishes the strength of the interstrand 3S-FlpC=O...H-NGly hydrogen bond. Thus, other forces can counter the benefits derived from the proper preorganization. Although (Pro-Pro-Gly)7 and (4S-Flp-4R-Flp-Gly)7 do not form stable homotrimeric helices, mixtures of these two peptides form stable heterotrimeric helices containing one (Pro-Pro-Gly)7 strand and two (4S-Flp-4R-Flp-Gly)7 strands. This stoichiometry can be understood by considering the cross sections of the two possible heterotrimeric helices. This unexpected finding portends the development of a "code" for the self-assembly of determinate triple helices from two or three strands.     

Peptoid oligomers with alpha-chiral, aromatic side chains: sequence requirements for the formation of stable peptoid helices.

The achiral backbone of oligo-N-substituted glycines or "peptoids" lacks hydrogen-bond donors, effectively preventing formation of the regular, intrachain hydrogen bonds that stabilize peptide alpha-helical structures. Yet, when peptoids are N-substituted with alpha-chiral, aromatic side chains, oligomers with as few as five residues form stable, chiral, polyproline-like helices in either organic or aqueous solution. The adoption of chiral secondary structure in peptoid oligomers is primarily driven by the steric influence of these bulky, chiral side chains. Interestingly, peptoid helices of this class exhibit intense circular dichroism (CD) spectra that closely resemble those of peptide alpha-helices. Here, we have taken advantage of this distinctive spectroscopic signature to investigate sequence-related factors that favor and disfavor stable formation of peptoid helices of this class, through a comparison of more than 30 different heterooligomers with mixed chiral and achiral side chains. For this family of peptoids, we observe that a composition of at least 50% alpha-chiral, aromatic residues is necessary for the formation of stable helical structure in hexameric sequences. Moreover, both CD and 1H-13C HSQC NMR studies reveal that these short peptoid helices are stabilized by the placement of an alpha-chiral, aromatic residue on the carboxy terminus. Additional stabilization can be provided by the presence of an "aromatic face" on the helix, which can be patterned by positioning aromatic residues with three-fold periodicity in the sequence. Extending heterooligomer chain length beyond 12-15 residues minimizes the impact of the placement, but not the percentage, of alpha-chiral aromatic side chains on overall helical stability. In light of these new data, we discuss implications for the design of helical, biomimetic peptoids based on this structural motif.     

Thermally induced conformation transition of triple-helical lentinan in NaCl aqueous solution

Lentinan, a beta-(1-->3)-D-glucan, was isolated from Lentinus edodes by using an improved extraction and purification method to show good water solubility and high yield. The results from 13C NMR, size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), dynamic light scattering (DLS), and optical rotation revealed that lentinan existed in a triple-helical conformation in the aqueous solution at 25 degrees C, whereas the thermally induced conformation transition from triple helix to single flexible chains occurred at elevated temperatures. The dependences of the weight-average molecular weight (Mw), radius of gyration (z1/2), hydrodynamic radius (Rh), intrinsic viscosity ([eta]), and specific optical rotation of lentinan on temperature in 0.9% NaCl aqueous solution showed an abrupt drop at 130-145 degrees C. It was confirmed that the conformation transitions from triple strand to single chain and from extended chains to winding chains for lentinan were completed rapidly at 130-145 degrees C, as a result of the simultaneous destruction of the intra- and intermolecular hydrogen bonds in lentinan. The thermally induced conformational transition was irreversible. The results from atomic force microscopy (AFM) and DLS demonstrated the existence of intrachain entanglement for the triple-helical chains, leading to the wormlike linear, circular, and crossover species for lentinan having high Mw (1.71x10(6)) in aqueous solution at 25 degrees C.     

Crowding effect on helix-coil transition: beyond entropic stabilization

We report circular dichroism measurements on the helix-coil transition of poly(L-glutamic acid) in solution with polyethylene glycol (PEG) as a crowding agent. The PEG solutions have been characterized by small angle neutron scattering and are well described by the picture of a network of mesh size ξ, usual for semi-dilute chains in good solvent. We show that the increase of PEG concentration stabilizes the helices and increases the transition temperature. But more unexpectedly, we also notice that the increase of concentration of crowding agent reduces the mean helix extent at the transition, or in other words reduces its cooperativity. This result cannot be taken into account for by an entropic stabilization mechanism. Comparing the mean length of helices at the transition and the mesh size of the PEG network, our results strongly suggest two regimes: helices shorter or longer than the mesh size.     

A triple helix of double helicates: three hierarchical levels of self-assembly in a single structure

The bis-bidentate bridging ligand L reacts with Ag(I) ions to form a conventional dinuclear [Ag(2)L(2)](2+) double helicate; individual double helicate units assemble via Ag···Ag interactions into infinite chains, three of which wrap around a central spine of anions to give a triple helical braid, which is therefore an infinite triple helix composed of molecular double helicate subunits.     

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.     

Physically and Chemically Crosslinked Gelatin Gels

In this paper, we examine the rheological and the structural properties of different types of gelatin networks, physical, chemical and both. The physical gel is due to the formation of collagen type triple helices when cooling the solutions. Chemically crosslinked gels are obtained with a reagent, in our case the bis(vinylsulfonyl)methane (BVSM), kindly provided by Kodak Industrie (France). Each BVSM molecule provides two covalent bonds. The chemical reaction was followed by microcalorimetry (MicroDSC III from Setaram, Caluire, France). The relation between shear moduli and crosslinking for the three types of gels is discussed, in relation with theoretical models of randomly crosslinked systems.