Biophysical functionality in polysaccharides: from Lego-blocks to nano-particles

The objective of the paper is to show the very important biophysical concepts that have been developed with polysaccharides. In particular, an attempt will be made to relate “a posteriori” the fundamental aspects, both experimental and theoretical, with some industrial applications of polysaccharide-based materials. The overview of chain conformational aspects includes relationships between topological features and local dynamics, exemplified for some naturally occurring carbohydrate polymers. Thus, by using simulation techniques and computational studies, the physicochemical properties of aqueous solutions of polysaccharides are interpreted. The relevance of conformational disorder–order transitions, chain aggregation, and phase separation to the underlying role of the ionic contribution to these processes is discussed. We stress the importance of combining information from analysis of experimental data with that from statistical–thermodynamic models for understanding the conformation, size, and functional stability of industrially important polysaccharides. The peculiar properties of polysaccharides in industrial applications are summarized for the particularly important example of nanoparticles production, a field of growing relevance and scientific interest.     

Conformational analysis of the tachykinins in solution: substance P and physalaemin.

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of beta-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.     

Conformational Analysis of the Tachykinins in Solution: Substance P and Physalaemin

Abstract

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of β-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.     

Circular dichroic properties and conformation of thionicotinamide dinucleotides.

The spectroscopic properties of the 3-thioamide analogues of coenzymes NAD and NADH (sNAD and sNADH) have been investigated in order to obtain information about their conformational properties. In particular, ultraviolet absorption and circular dichroism properties of solutions in phosphate buffer pH 7 and ethanol were studied. Also equimolar mixtures of AMP and sNMN(H), obtained by cleaving the coenzymes with phosphodiesterase, were investigated using the same solvents. The appearance of a couplet around 260 nm, which is not present for the mixture of sNMN and AMP, suggests a stacking interaction of the two aromatic moieties in sNAD. This conclusion is further substantiated by a hyperchroism of the ultraviolet absorption band in the 260-nm region in both sNAD and sNADH. The comparison of the ultraviolet and circular dichroic properties of intact and cleaved coenzymes in the different solvent systems makes it possible to single out the bands which are more sensitive to conformation changes (i.e. to open-stacking equilibrium) and those which are sensitive to solvent effects only.     

Novel genipin-cross-linked chitosan/silk fibroin sponges for cartilage engineering strategies

The positive interaction of materials with tissues is an important step in regenerative medicine strategies. Hydrogels that are obtained from polysaccharides and proteins are expected to mimic the natural cartilage environment and thus provide an optimum milleu for tissue growth and regeneration. In this work, novel hydrogels composed of blends of chitosan and Bombyx mori silk fibroin were cross-linked with genipin (G) and were freeze dried to obtain chitosan/silk (CSG) sponges. CSG sponges possess stable and ordered structures because of protein conformational changes from alpha-helix/random-coil to beta-sheet structure, distinct surface morphologies, and pH/swelling dependence at pH 3, 7.4, and 9. We investigated the cytotoxicity of CSG sponge extracts by using L929 fibroblast-like cells. Furthermore, we cultured ATDC5 cells onto the sponges to evaluate the CSG sponges' potential in cartilage repair strategies. These novel sponges promoted adhesion, proliferation, and matrix production of chondrocyte-like cells. Sponges' intrinsic properties and biological results suggest that CSG sponges may be potential candidates for cartilage tissue engineering (TE) strategies.     

Photomodulation of conformational states. IV. Integrin-binding RGD-peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent

In previous studies we have investigated octapeptides backbone-cyclized by (4-amino)phenyl azobenzoic acid (APB) or (4-aminomethyl)phenylazobenzoic acid (AMPB) and containing the active-site sequence Cys-Ala-Thr-Cys-Asp from the thioredoxin reductase. The conformational and redox properties of these peptides were strongly dependent on the isomeric state of the azobenzene chromophore. Using the same approach we were successful in constructing photoresponsive ligands for alphavbeta3 integrin containing the Arg-Gly-Asp (RGD) sequence as binding motif. For achieving maximal conformational restriction of the peptide a reduced ring size compared to our previous azobenzene peptides was employed in the cyclic peptide c[Asp-D-Phe-Val-AMPB-Lys-Ala-Arg-Gly-]. Conformational properties of the trans and cis isomers of this peptide in solution were investigated by CD and NMR and were found to differ markedly from the thioredoxin derived azobenzene peptides. In a second peptide, c[Asp-D-Phe-Val-Lys-AMPB-Ala-Arg-Gly-], shifting the position of the chromophore lead to a marked decrease in affinity. With the availability of the x-ray structure of a cyclic RGD-pentapeptide bound to alphavbeta3 integrin (PDB entry 1L5G) modeling of possible bound conformations for trans and cis isomers of both azobenzene peptides was possible. Notably, both peptides in either isomeric form share the same overall conformation in the bound state according to our molecular dynamics simulations.     

Conformational studies on somatostatin. II. The C-terminal hexapeptide fragment

The results of a conformational study on the C terminal hexapeptide of Somatostatin are presented. Semi-empirical energy calculations and high resolution NMR methods have been used to obtain information on the conformational properties of SRIF9-14 in [2H6]dimethylsulfoxide and 2H2O. It is concluded from the energy calculations that the peptide has an averaged conformation in which semi extended and folded structures are important. Only some of the folded conformations can explain the chemical shift differences between the amino acid residues Thr10 and Thr12 as a ring current shift by the Phe11 aromatic ring on Thr10. The nonequivalence is more pronounced in dimethyl-sulfoxide (0.23--0.15 ppm) where it decreases with increasing temperature towards the temperature independent value in 2H2O (0.03 ppm). This suggests that the folded conformations are somewhat predominant in dimethylsulfoxide solutions. In 2H2O the semi extended and folded structures are statistically equally important and the peptide is more flexible. A comparison with a study on the smaller fragments SRIF10-12 and SRIF10-13 which have similar conformational properties, demonstrates the usefulness of the fragment approach in conformational studies of peptides.     

Controlled sulfatation of natural anionic bacterial polysaccharides can yield agents with specific regenerating activity in vivo

The regenerating activities of chemically modified anionic bacterial polysaccharides by O-sulfonation were investigated using a in vivo model of rat injured muscle regeneration. Glucuronan (GA), a linear homopolysaccharide of -->4)-beta-D-GlcpA-(1--> residues partially acetylated at the C-3 and/or the C-2 position, and glucoglucuronan (GGA), a linear heteropolysaccharide of -->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1--> residues were sulfated. SO3-DMF sulfatation complex provided polysaccharides with different sulfur contents, however, a depolymerization occurred because we did not use large excess of pyridine to obtain pure modified polysaccharides. A regenerating activity on injured extensor digitorum longus (EDL) muscles on rats was obtained with these two sulfated anionic polymers. The position of sulfate groups on glucoglucuronan (primary or secondary alcohol) seems to have no influence on the biological activity by opposition to the degree of sulfatation both for the glucuronans and the glucoglucuronans. The yield of acetate groups in the glucuronan polymer modulated the specific activity.     

Spectroscopic characterisation of order-disorder transitions for extracellular polysaccharides of arthrobacter species

The conformational behaviour of the extracellular polysaccharides from Arthrobacter species of soil-borne bacteria has been investigated by nuclear magnetic resonance relaxation and optical rotation. Polysaccharides from A. stabilis, A. viscosus, and A. viscosus sp. n, in solution at room temperature, all show evidence of an ordered conformation which can be melted out on heating. The temperature course of this transition, however, shows considerable variation with bacterial species. Thus A. stabilis polysaccharide shows a very sharp conformational transition centred around 60°, whereas the transitions of the polysaccharides from both strains of A. viscosus occur over a much broader temperature-range. The transition for the polysaccharide of A. viscosus sp. n is again centred close to 60°, whereas, for A. viscosus, melting of the tertiary structure of the polysaccharide is incomplete at 100°. O-Deacetylation destroys the ordered conformation of both A. viscosus polysaccharides. The ordered structure of A. stabilis polysaccharide, by contrast, is stabilised by removal of acyl substituents (which here include succinic half-ester). Understanding of the conformational state of these materials affords considerable insight into their gelation behaviour and unusual solution rheology. The known solution interactions with certain plant polysaccharides suggest a possible biological role for Arthrobacter polysaccharides in relationships with components of plant root-systems.     

Ciliary dynein conformational changes as evidenced by the extrinsic fluorescent probe 8-anilino-1-naphthalenesulfonate

The binding of 8-anilino-1-naphthalenesulfonate (ANS) to ciliary dynein ATPase leads to a marked increase in the dye's fluorescence intensity, accompanied by a blue shift in the observed fluorescence emission maximum. We found that dynein has 37 +/- 3 ANS binding sites and that experimentally applied ANS concentrations failed to alter enzyme activity. The fluorescence properties of the enzyme-dye complex were used to learn more about the binding characteristics of dynein substrates and effectors and to probe for possible conformational changes of the enzyme. The fluorescence of the dynein-ANS complex is increased by a number of substrates, including ATP, GTP, and UTP. The transfer of excitation energy from dynein chromophores to adsorbed ANS was also investigated. Our findings indicate that dynein appears to undergo a localized conformational change in its interaction with ATP. Native dynein was also found to be conformationally different from heat-activated or NEM-modified enzyme as evidenced by the emission and excitation spectra of the various enzyme-ANS complexes.     

Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA

Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K(+) channel KcsA under near-physiological conditions, using solution-state NMR. In a series of (1)H(15)N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K(+) channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a "pH-sensor" for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed "dual gating" properties of the K(+) channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.     

Does 13C-or 15N-labeling affect Cu(I)-thiolate cluster arrangement in yeast copper-metallothionein?

It was attempted to examine whether or not isotope labeling may possibly affect an oligonuclear metal-thiolate cluster. Cu-metallothioneins are known to contain strongly distorted Cu-thiolate clusters and seemed appropriate for this study. Thus, yeast ¹³C-and ¹⁵N-Cu-metallothioneins were isolated from Saccharomyces cerevisiae cells grown in a minimal synthetic medium and some physicochemical parameters were compared with those of the unlabeled Cu-thionein. Surprisingly, the ¹³C- and ¹⁵N- labeled Cu₇-thioneins are distinctly different in their characteristic spectroscopic properties. The electronic absorption was blue-shifted while both luminescence emission and chiroptic features display a distinct red shift with markedly diminished intensities, respectively. Contrary to common knowledge that isotope labeling does not affect the molecular architecture of a protein the present results support such a phenomenon. Attributable to the fortunate happenstance that there is a strongly distorted structural situation in the oligonuclear Cu-thiolate cluster this isotope effect came to light.     

Structural changes in the NH2-terminal domain of fibronectin upon interaction with heparin. Relationship to matrix-driven translocation

The effects of heparin and various related polysaccharides on the circular dichroic spectra of fibronectin and its 31-kDa NH2-terminal tryptic fragment were studied. These effects were evaluated with respect to (i) spectral features of the native proteins that are sensitive to pH denaturation and breaking of disulfide bonds, (ii) sensitivity of spectral changes to Ca2+, and (iii) the fibronectin-dependent interfacial interaction known as "matrix-driven translocation." We found that native heparin causes an attenuation of the positive CD peak at 228 nm with both the intact protein and the fragment, and causes a small but reproducible red shift in the spectrum of the fragment. All of these changes are analogous to spectral changes seen with denaturation or reduction of the proteins. In contrast to the situation with the intact protein, the heparin-induced spectral changes in the fragment were abolished in the presence of 10 mM Ca2+. Desulfation of heparin lessened or destroyed its ability to induce these changes, and carboxymethylated heparin and dextran sulfate induced different kinds of spectral alterations. Fibronectin and heparin determinants required for the induction of the characteristic spectral shift of the NH2-terminal domain corresponded to those required for matrix-driven translocation, suggesting that the associated conformational change in fibronectin plays a role in this biophysical effect.     

Use of 13C chemical shift surfaces in the study of carbohydrate conformation. Application to cyclomaltooligosaccharides (cyclodextrins) in the solid state and in solution

The anomeric carbon chemical shifts of free cyclomaltohexaose, -heptaose, -octaose, -decaose, and -tetradecaose (alpha-, beta-, gamma-, epsilon-, and eta-cyclodextrin, respectively), and of alpha-cyclodextrin inclusion complexes, both in the solid state and in solution, were computed using ab initio 13C chemical shift surfaces for the D-Glcp-alpha-(1-->4)-D-Glcp linkage as a function of the glycosidic bond dihedral angles. Chemical shift calculations in the solid state used angle pairs measured from cyclodextrin X-ray structures as input. For estimations in the liquid state two different approaches were employed to account for dynamic averaging. In one, the computed solid-state anomeric carbon chemical shifts for each cyclodextrin D-Glcp monomer were simply averaged to obtain an estimate of the 13C shifts in solution. In the other, chemical shifts for the anomeric carbons were determined by averaging back-calculated 13C shift trajectories derived from a series of 5 ns molecular dynamic simulations for the oligosaccharides with explicit representation of water. Good agreement between calculated and experimental 13C shifts was found in all cases. Furthermore, our results show that the ab initio 13C chemical shift surfaces are sufficiently sensitive to reproduce the small variations observed for the anomeric 13C shifts of the different cyclodextrin D-Glcp units in the solid state with excellent accuracy. The use of chemical shift surfaces as tools in conformational studies of oligosaccharides is discussed.     

Molar mass distribution of hydroxypropyl cellulose by size exclusion chromatography with dual light scattering and refractometric detection

Physico-chemical properties of cellulose derivatives are of considerable interest in many technical applications, for example, in the food and drug industry. Efficient and careful characterisation of these properties is thus highly desirable. In this study, two different size exclusion chromatography (SEC) systems, connected on-line either to a low-angle laser light scattering detector (LALLS) or to a multi-angle laser light scattering detector (MALLS), are employed for size characterisation of three batches of hydroxypropyl cellulose (HPC) from different manufacturers. All three samples turned out to have a weight average molar mass around 100,000 g/mol, but considerable differences concerning conformational properties were found. Two of the samples contained compact components, presumably aggregates of HPC, which were clearly detected by both SEC-systems. The third sample, obtained from another manufacturer, did not show any indication of aggregation. Both SEC-MALLS and SEC-LALLS are proven to be efficient techniques for characterisation of complex polysaccharides like HPC containing mixtures of solvated polymer chains, as well as micelle-like aggregates.     

Floc destruction and its impact on dewatering properties in the process of using flat-sheet membrane for simultaneous thickening and digestion of waste activated sludge

Floc destruction and its impacts on the dewatering properties in terms of capillary suction time (CST) in the process of using flat-sheet membrane for simultaneous thickening and digestion (MSTD) of waste activated sludge were investigated. A pilot-scale MSTD reactor was used to treat waste activated sludge, and extracellular polymeric substances (EPS), soluble biopolymers, dissolved cations and the dewatering properties were measured. The results indicated that the destruction mechanisms of the MSTD process could be divided into two phases due to the variation of dissolved oxygen (DO). Polysaccharides and proteins were released in both phases, but the release patterns were different. The concentration of polysaccharides was much greater than that of proteins in Phase 1, while the ratio of proteins to polysaccharides ranged from 1.5 to 1.7. The divalent cations in supernatant significantly increased in Phase 1, while the monovalent cations in supernatant rose in Phase 2. The dewatering properties in terms of CST significantly increased in the MSTD process, and the mixed liquor suspended solids (MLSS), particle size, and biopolymers in supernatant had significant effects on the dewatering properties.     

Study of conformational changes in immunoglobulin M using the method of differential spectroscopy

Using differential and solvent-perturbation spectrophotometry, the nature of conformational changes in immunoglobulin M (IgM) in different regimens was investigated. The quantities of tryptophan and tyrosine chromophores exposed on the surface of the molecule and screened, were evaluated. The changes in pH (7.8----2.0) of the surrounding medium and splitting of carbohydrate groups from IgM were shown to cause opposite effects, i. e., a "blue shift" of the spectrum and exposure of new chromophores by acidification, and a "red shift" and screening of chromophores by splitting of carbohydrate groups. The experimental results agree well with the previously made assumption on the differences in the spatial conformational changes in the IgM molecule under effects of pH of the surrounding medium and the loss of carbohydrate groups. Analysis of the spectral characteristics of some free Fab- and (Fc)5-fragments derived from the IgM molecules allowed a specification of the changes occurring in different parts of the whole molecule. The main conformational changes after acidification occur in the (Fc)5-fragment responsible for the effector function of IgM.     

The family 52 β-xylosidase from Geobacillus stearothermophilus is a dimer: Structural and biophysical characterization of a glycoside hydrolase

Xylans are the most abundant polysaccharides forming the plant cell wall hemicelluloses, and they are degraded, among other proteins, by β-xylosidase enzymes. In this work, the structural and biophysical properties of the family 52 β-xylosidase from Geobacillus stearothermophilus, XynB2, are described. Size exclusion chromatography, analytical centrifugation, ITC, CD, fluorescence (steady state and ANS-binding) and FTIR were used to obtain the structure, the oligomerization state and the conformational changes of XynB2, as pH, chemical denaturants or temperature were modified. This report describes the first extensive conformational characterization of a family 52 β-xylosidase. The active protein was a highly hydrated dimer, whose active site was formed by the two protomers, and it probably involved aromatic residues. At low pH, the protein was not active and it populated a monomeric molten-globule-like species, which had a conformational transition with a pK_a of ~ 4.0. Thermal and chemical-denaturations of the native protein showed hysteresis behaviour. The protein at physiological pH was formed by α-helix (30%) and β-sheet (30%), as shown by CD and FTIR. Comparison with other xylosidases of the same family indicates that the percentages of secondary structure seem to be conserved among the members of the family.     

Perturbation of the conformational equilibria in Ras by selective mutations as studied by 31P NMR spectroscopy

Ras regulates a variety of different signal transduction pathways acting as molecular switch. It was shown by liquid and solid-state (31)P NMR spectroscopy that Ras exists in the guanosine-5'-(beta,gamma-imido)triphosphate bound form in at least two conformational states interconverting in millisecond time scale. The relative population between the two conformational states affects drastically the affinity of Ras to its effectors. (31)P NMR spectroscopy shows that the conformational equilibrium can be shifted specifically by point mutations, including mutations with oncogenic potential, thus modifying the effector interactions and their coupling to dynamic properties of the protein.     

The family 52 beta-xylosidase from Geobacillus stearothermophilus is a dimer: structural and biophysical characterization of a glycoside hydrolase

Xylans are the most abundant polysaccharides forming the plant cell wall hemicelluloses, and they are degraded, among other proteins, by beta-xylosidase enzymes. In this work, the structural and biophysical properties of the family 52 beta-xylosidase from Geobacillus stearothermophilus, XynB2, are described. Size exclusion chromatography, analytical centrifugation, ITC, CD, fluorescence (steady state and ANS-binding) and FTIR were used to obtain the structure, the oligomerization state and the conformational changes of XynB2, as pH, chemical denaturants or temperature were modified. This report describes the first extensive conformational characterization of a family 52 beta-xylosidase. The active protein was a highly hydrated dimer, whose active site was formed by the two protomers, and it probably involved aromatic residues. At low pH, the protein was not active and it populated a monomeric molten-globule-like species, which had a conformational transition with a pK(a) of approximately 4.0. Thermal and chemical-denaturations of the native protein showed hysteresis behaviour. The protein at physiological pH was formed by alpha-helix (30%) and beta-sheet (30%), as shown by CD and FTIR. Comparison with other xylosidases of the same family indicates that the percentages of secondary structure seem to be conserved among the members of the family.