Helix stabilization of poly(ethylene glycol)-peptide conjugates

Hybrid polymer-peptide conjugates offer the potential for incorporating biological function into synthetic materials. The secondary structure of short helical peptides, however, frequently becomes less stable when expressed independent of longer protein sequences or covalently linked with a conformationally disordered synthetic polymer. Recently, new amphipathic peptide-poly(ethylene glycol) conjugates were introduced (Shu, J., et al. Biomacromolecules 2008, 9, 2011), which displayed enhanced peptide helicity upon polymer functionalization while retaining tertiary coiled-coil associations. We report here a molecular simulation study of peptide helix stabilization by conjugation with poly(ethylene glycol). The polymer oxygens are shown to favorably interact with the cationic lysine side chains, providing an alternate binding site that protects against disruption of the peptide hydrogen-bonds that stabilize the helical conformation. When the peptide lysine charges are neutralized or poly(ethylene glycol) is conjugated with polyalanine, the polymer exhibits a negligible effect on the secondary structure. We also observe the interactions of poly(ethylene glycol) with the amphipathic peptide lysines tends to segregate the polymer away from the nonpolar face of the helix, suggesting no disruption of the interactions that drive tertiary contacts between helicies.     

A new bacterial exopolysaccharide (YAS34). II. Influence of thermal treatments on the conformation and structure. Relation with gelation ability

This paper describes an original mechanism of evolution of a polysaccharide system occurring during thermal treatments. Under its native conformation, the YAS34 polymer presents a solution character in the dilute and semi-dilute regimes. However, the zero shear rate viscosity indicates existence of interchain interactions which disappear on the deacetylated polymer. Thermal treatments over the temperature of conformational change produce a progressive and irreversible evolution of the physical properties when the polymer is under its sodium salt form. This evolution was related to a modification of the arrangement of acetyl substituents. The heated polysaccharide gives thermoreversible gel which is very elastic. A gelation mechanism is proposed based on formation of helical segments connecting the network.     

Distinct gelation mechanism between linear and branched (1--3)- beta-D-glucans as revealed by high resolution solid state 13C NMR

We have recorded high-resolution 13C-NMR spectra of linear (curdlan) and branched (lentinan, HA-beta-glucan and its polyol and aldehyde derivatives) (1----3)-beta-D-glucans in hydrate and gel states, in order to gain insight into their gelation mechanism. Network structure of curdlan turned out to be highly heterogeneous from its motional state, from liquid-like, through intermediate, to solid-like domains. They are studied by a variety of experiments, conventional high-resolution NMR by broad-band decoupling, high-power decoupling with magic angle spinning (MAS), and cross-polarization-magic-angle-spinning (CP-MAS). Nevertheless, we found that conformations of these distinct liquid-like and solid-like domains exhibit an identical single helix conformation with a small proportion of a triple helix form, supporting our previous view as to the gelation mechanism. In contrast, the network structure of branched (1----3)-beta-D-glucans in the gel state arises mainly from the triple helix conformation. This means that gelation of branched (1----3)-beta-D-glucan proceeds from partial association of the triple helical chains, previously proposed for gelation of a linear glucan. Furthermore, we found that conversion from the single chain to the single helix was not achieved readily by hydration of over 8 h at 96% R.H. for branched glucan but the triple helix form is obtained when these samples are hydrated fully as in gel state.     

Structural basis for dimerization of the Dictyostelium gelation factor (ABP120) rod.

Gelation factor (ABP120) is one of the principal actin-cross-linking proteins of Dictyostelium discoideum. The extended molecule has an N-terminal 250-residue actin-binding domain and a rod constructed from six 100-residue repeats that have an Ig fold. The ability to dimerize is crucial to the actin cross-linking function of gelation factor and is mediated by the rod in which the two chains are arranged in an antiparallel fashion. We report the 2.2 A resolution crystal structure of rod domains 5 and 6, which shows that dimerization is mediated primarily by rod domain 6 and is the result of a double edge-to-edge extension of beta-sheets. Thus, contrary to earlier proposals, the chains of the dimeric gelation factor molecule overlap only within domain 6, and domains 1-5 do not pair with domains from the other chain. This information allows construction of a model of the gelation factor molecule and suggests how the chains in the related molecule filamin (ABP280) may interact.     

Mesoscopic gel at low agarose concentration in water: a dynamic light scattering study.

Previous work in our laboratory has shown that at very low agarose concentration in water gelation still occurs within mutually disconnected, high concentration regions generated by spinodal demixing. The freely diffusing particles obtained in these conditions are studied in the present work by depolarized dynamic light scattering and probe diffusion experiments. These particles are found to behave as large (in fact, mesoscopic) polymer fibers entangled in a continuously rearranged mesh with scaling parameters typical of partially flexible, neutral chains. The present results allow specifying the notion of mesoscopic gelation. They also reveal that the same symmetry-breaking mechanism that allows macroscopic gelation at polymer concentrations well below the threshold for random cross-link percolation generates additional and unexpected phenomena.     

Structural characterisation of thermoreversible anionic polysaccharide gels by their elastoviscous properties

Previously reported results obtained for the elastoviscous properties of some thermoreversible gels formed from anionic polysaccharides (high methoxyl pectin, furcellaran and κ-carrageenan) and also gelatin and maltodextrin are discussed and some conclusions about the structure of the gels are presented.The rate at which the relaxation processes take place in the gel is independent of the polymer concentration suggesting that the gels are structurally inhomogeneous.If the helical conformation of the individual macromolecule is stable the standard enthalpy change on crosslink breakdown is less than 45 kJ mol^?1. A relatively small decrease in standard enthalpy is sufficient for network stability because of the low standard entropy loss on gelation which is typical of semi-rigid chain polymers. If, however, the helical conformation is unstable the gelation process is cooperative and the standard enthalpy change on crosslink breakdown exceeds 200 kJ mol^?1.     

Helical Inversion of Gel Fibrils by Elongation of Perfluoroalkyl Chains as Studied by Vibrational Circular Dichroism

Vibrational circular dichroism (VCD) spectroscopy was applied to gelation by a chiral low‐molecular mass weight gelator, N,N’‐diperfluoroalkanoyl‐1,2‐trans‐diaminocyclohexane. Attention was focused on the winding effects of (–CF₂)_n chains on the gelating ability. For this purpose, a series of gelators were synthesized with perfluoroalkyl chains of different length (n = 6–8). When gelation was studied using acetonitrile as a solvent, the fibrils took different morphologies, depending on the chain length: twisted saddle‐like ribbon or helical ribbon from fibril (n = 6) and a helical ribbon from platelet (n = 8). The signs of VCD peaks assigned to the couplet of C=O stretching and to the C‐F stretching were also dependent on n, indicating that a gelator molecule changed conformation on elongating perfluoroalkyl chains. A model is proposed for the aggregation modes in fibrils. Chirality 28:361–364, 2016. © 2016 Wiley Periodicals, Inc.     

Influence of chain length and polymer concentration on the gelation of (amidated) low-methoxyl pectin induced by calcium

The gelation of low-methoxyl pectin (LMP) induced by addition of Ca2+ was studied by measuring the storage modulus as a function of temperature during cooling. Samples with different molar masses were prepared by mechanical degradation. The effect of the molar mass and the pectin concentration on the gelation properties was investigated. The effect of partial amidation was studied by comparing LMP and partially amidated LMP with the same molar mass and degree of methylation. The results are compared to those from a model developed for Ca2+-induced pectin gelation, and good agreement is found except at low concentrations and low molar masses where the gels are weaker than predicted. At low concentrations intrachain bonding weakens the gel, while the presence of small pectin chains weakens the gel because it neutralizes binding sites on larger chains.     

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA): its conformational and photodynamic properties

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA) polymers with 22 and 35 mol % of azo chromophores in the side chains have been synthesized by condensing 4-methoxy-4'-aminoazobenzene and poly(l-glutamic acid). These polymers have been characterized by NMR, FT-IR, and UV-visible spectroscopic techniques. The conformational features of the polymer backbone chains in the films that were cast from the polymer solutions prepared in different solvents have been investigated by circular dichroism spectroscopy. Experimental data suggested that the thermal cis-trans relaxation and photoinduced birefringence, which are related to the azo chromophores in the side chains of polymer, are not affected by the conformations of polymer backbones. However, the modulations of the surface relief gratings, the result of photoinduced mass transport process, recorded on these polymers are sensitive to polymer main chain conformation, as well as the degree of functionalization.     

Plausible molecular and crystal structures of chitosan/HI type II salt

Chitosan/HI type II salt prepared from crab tendon was investigated by X-ray fiber diffraction. Two polymer chains and 16 iodide ions (I(-)) crystallized in a tetragonal unit cell with lattice parameters of a = b = 10.68(3), c (fiber axis) = 40.77(13) A, and a space group P4(1). Chitosan forms a fourfold helix with a 40.77 A fiber period having a disaccharide as the helical asymmetric unit. One of the O-3... O-5 intramolecular hydrogen bonds at the glycosidic linkage is weakened by interacting with iodide ions, which seems to cause the polymer to take the 4/1-helical symmetry rather than the extended 2/1-helix. The plausible orientations of two O-6 atoms in the helical asymmetric unit were found to be gt and gg. Two chains are running through at the corner and the center of the unit cell along the c-axis. They are linked by hydrogen bonds between N-21 and O-61 atoms. Two out of four independent iodide ions are packed between the corner chains while the other two are packed between the corner and center chains when viewing through the ab-plane. The crystal structure of the salt is stabilized by hydrogen bonds between these iodide ions and N-21, N-22, O-32, O-61, O-62 of the polymer chains.     

Diaspirins that cross-link beta chains of hemoglobin: bis(3,5-dibromosalicyl) succinate and bis(3,5-dibromosalicyl) fumarate.

Two double-headed aspirins, bis(3,5-dibromosalicyl) succinate and bis(3,5-dibromosalicyl) fumarate, have been found to be potent acylating agents of intracellular hemoglobin (A or S) in vitro. Furthermore, each of these reagents cross-links beta chains of hemoglobin, probably at the beta cleft. The modified hemoglobins show increased oxygen affinities and reduced gelation or sickling tendencies.     

The effect of glycosaminoglycans (GAG) on the intramolecular bindings of collagen.

The subfractions of collagen (alpha, beta and gamma components) precipitated from neutral collagen solution by gelation corresponded to those of collagen precipitated in the same conditions (ion environments) and in the presence of chondroitin-4-sulphate. Collagen fibrils precipitated delayed in the presence of heparin poor in beta chains and rich in alpha chains. The delaying effect of oversulphated GAG on the in vitro fibril formation can be explained by the release or prevention of ion-type intramolecular bonds of collagen. The possible in vivo significance of the above bonds is discussed.     

Chondrogenic properties of human periosteum-derived progenitor cells (PDPCs) embedded in a thermoreversible gelation polymer (TGP)

Periosteum-derived progenitor cells (PDPCs) were isolated using a fluorescence-activated cell sorter and their chondrogenic potential in biomaterials was investigated for the treatment of defective articular cartilage as a cell therapy. The chondrogenesis of PDPCs was conducted in a thermoreversible gelation polymer (TGP), which is a block copolymer composed of temperature-responsive polymer blocks such as poly(N-isopropylacrylamide) and of hydrophilic polymer blocks such as polyethylene oxide, and a defined medium that contained transforming growth factor-β3 (TGF-β3). The PDPCs exhibited chondrogenic potential when cultured in TGP. As the PDPCs-TGP is an acceptable biocompatible complex appropriate for injection into humans, this product might be readily applied to minimize invasion in a defected knee.     

Noncovalent adducts of poly(ethylene glycols) with proteins

A new method of preparation of noncovalent complexes between poly(ethylene glycol) (PEG) and proteins (alpha-chymotrypsin (ChT), lysozyme, bovine serum albumine) under high pressure has been developed. The involvement of polymer in the complexes was proved using (3)H-labeled PEG. The composition of the complexes (the number of polymer chains per one ChT molecule) depends on the molecular mass of PEG and decreases with the increase in molecular mass from 300 to 4000, whereas the portion of the protein (wt %) in complexes does not depend on the molecular mass of incorporated PEG and corresponds to approximately 70 wt %. The kinetic constants for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester and azocasein catalyzed by the PEG-ChT complexes are identical with the corresponding values for the native ChT. According to the data obtained by the method of circular dichroism, the enzyme in the complexes fully retains its secondary structure. The steric availability of PEG polymer chains in the complexes was evaluated by their complexation with alpha-cyclodextrin (CyD) or polymer derivatives of beta-CyD modified with PEG (PEG-beta-CyD). In contrast to free PEG, only part of PEG polymer chains ( approximately 10%) interact with alpha-CyD. Thus, the complexation of PEG with ChT proceeds by means of multipoint interaction with surface groups of the protein globule located far from the active site and results in the sufficient decrease in the availability of polymer chains. The complexes between PEG chains in PEG-protein adducts and PEG-beta-CyD may be considered as a novel type of dendritic structures.     

Basement membrane (type IV) collagen is a heteropolymer

Type IV collagen was isolated in high yield from bovine kidney cortex. The protein revealed Mr = 380,000 and contained, in a 2:1 ratio, two different disulfide-linked polypeptide chains, C-1 and D-1 (Mr = 125,000). Carboxymethyl-cellulose chromatography before and after reduction proved that the two polypeptide chains are arranged in a single triple helical molecule with the chain composition (C-1)2(D-1). The disulfide bridges appear to be located 180 amino acid residues from the NH2 terminus of the chains.     

The Specificity of Antibodies Elicited by Poly(dG)-Poly(dC)

Abstract

Antibodies have been raised to the synthetic DNA polymer poly(dG)·poly(dC). These antibodies have the ability to distinguish this right-handed polymer from natural mixed sequence DNA, as well as from other right- and left-handed synthetic DNA polymers. They show reduced but measurable binding to synthetic polymers which contain various combinations of guanine and cytosine polynucleotides suggesting that both helical shape and sequence are recognized by this antiserum.     

The spatial configuration of ordered polynucleotide chains. I. Helix formation and base stacking.

A single virtual bond scheme set forth previously for the treatment of average properties of randomly coiling polynucleotides is here applied to the calculation of helical parameters which characterize a regularly repeating polynucleotide molecule. Only a fraction of the enormous number of conformationally feasible helixes fulfill the geometric criteria of vertical base stacking usually associated with ordered polynucleotide chains. Detailed examination of the nature and mode of base stacking feasible in a single helical backbone structure indicates that the handedness of a base stacking arrangement does not correlate either quantitatively or qualitatively with the handedness of the polymer backbone. A number of polynucleotide chains which exhibit lefthanded base stacking patterns in nmr and CD studies may, in fact, be righthanded helixes.     

Chirality responsive helical poly(phenylacetylene) bearing L-proline pendants

A novel, optically active, cis-transoidal poly(phenylacetylene) bearing an L-proline residue as the pendant group (poly-1) was prepared by the polymerization of the corresponding monomer using a rhodium catalyst in water, and its chiroptical property was investigated using circular dichroism spectroscopy. Poly-1 showed intense Cotton effects in the UV-visible region of the polymer backbone in water, resulting from the prevailing one-handed helical conformation induced by the covalent-bonded chiral L-proline pendants and exhibited a unique helix-sense inversion in response to external, achiral, and chiral stimuli, such as the solvent and interactions with chiral small molecules. We found that poly-1 could enantioselectively trap 1,1'-2-binaphthol within its hydrophobic helical cavity inside the polymer in aqueous media and underwent an inversion of its helical sense in the presence of one of the enantiomers. The effect of the optical purity of 1,1'-2-binaphthol on the chiroptical properties of poly-1 was also investigated.     

Adsorption of polypeptides on solid surfaces. I. Effect of chain stiffness

The general method of obtaining the partition function and thermodynamic characteristics of polymer chains near an adsorbing surface, simulated by random walks on a lattice, is developed. The method takes into account the effect of short-range interactions in polymer chains, in particular, the chain stiffness and secondary structure. The theory of adsorption of chains of different stiffness is developed, and the process of adsorption which occurs when the external conditions change is shown to be always a second-order phase transition. The critical adsorption energy decreases and the sharpness of transition grows when the chain stiffness increases. A simple model of a chain with “virtual” steps is proposed which simplifies the treatment; the results obtained are in good agreement with exact theories. A general scheme of analysis of adsorption of chains with a given secondary structure is set forth and the analogy between the stiffness of a noncooperative chain and the presence of helical segments in a polypeptide chain is discussed.     

Preparation and structural analysis of actinidain-processed atelocollagen of yellowfin tuna (Thunnus albacares)

Pepsin-hydrolyzed collagen (atelocollagen) is a trimer, consisting of alpha 1 and alpha 2 monomers, and shows molecular species corresponding to a monomer, dimer (beta chain), and trimer (gamma chain) by SDS-polyacrylamide gel electrophoresis. Atelocollagen was purified from yellowfin tuna (Thunnus albacares) by salt precipitation and cation-exchange chromatography. Enzymatic hydrolysis of the atelocollagen by actinidain, a cysteine protease purified from kiwifruit, was analyzed by SDS-polyacrylamide gel electrophoresis. The triple helical structure unique to collagen was retained in the atelocollagen as judged by circular dichroism spectra. The actinidain-processed atelocollagen showed only monomeric alpha 1 and alpha 2 chains, with no beta and gamma chains, by SDS-polyacrylamide gel electrophoresis; nevertheless, it retained the typical triple helical structure. It is suggested that actinidain cleaved the atelocollagen molecule at specific sites on the inside of the inter-strand cross-linking peptides.