Interrogation of single synthetic polymer chains and polysaccharides by AFM-based force spectroscopy.

This contribution reviews selected mechanical experiments on individual flexible macromolecules using single-molecule force spectroscopy (SMFS) based on atomic force microscopy. Focus is placed on the analysis of elasticity and conformational changes in single polymer chains upon variation of the external environment, as well as on conformational changes induced by the mechanical stress applied to individual macromolecular chains. Various experimental strategies regarding single-molecule manipulation and SMFS testing are discussed, as is theoretical analysis through single-chain elasticity models derived from statistical mechanics. Moreover, a complete record, reported to date, of the parameters obtained when applying the models to fit experimental results on synthetic polymers and polysaccharides is presented.     

A general treatment of the configuration statistics of polysaccharides.

A treatment of the configurational statistics of polysaccharides is given in the isomeric state approximation. All classes of linear polysaccharides of specifiec chemical sequence are treated simultaneously. Chain tortuosity arising from torsional motions about the chemical bonds of the glycosidic linkages is recognized explicitly as is the possibility for conformational isomerism of the sugar residues. Valence angles and lengths are taken to be fixed at the equilibrium values, and pyranose residues in their chair conformations are treated as inflexible constituents of the skeletal structure. Pyranose and furanose forms capable of pseudorotation may be incorporated as rigid skeletal entities as well, provided suitable attention is given to the selection and interpretation of the conformational isomeric states included. Separation of the configuration energy into independent contributions is shown to be impossible in general. Methods are described for assessing the influence of neighbor interactions on the populations of the several conformers of the sugar residues. The relative conformational free energy of the flexible and chair form conformers of pyranose sugars is discussed, and appropriate measures of polysaccharide chain flexibility and stiffness are suggested.     

Gas phase and water solution conformational analysis of the herbicide diuron (DCMU): an ab initio study

In the present work, the conformational equilibrium for the herbicide diuron (DCMU) has been investigated using high level ab initio calculations. The solvent effect was included through two different continuum models: (1) the real cavity IPCM method and (2) the standard dipole Onsager model SCRF. The effect due to solute-solvent hydrogen-bond interactions was analyzed considering a hybrid discreet-continuum model. At the Hartree-Fock level, the gas phase results showed that only the trans forms (A and B) are present in the equilibrium mixture, with the relative concentrations found to be 33% (A) and 67% (B) (HF/6-311+G**//6-31G**). When the electronic correlation effect is included (MP2/6-31G*//HF/6-31G*), a relative stabilization of the cis forms was observed, with the conformational distribution calculated as 38% (A), 50% (B), 6% (C) and 6% (D). The trans conformations were found to be completely planar, which has been considered to be a prerequisite for the herbicide binding. In water solution, the trans conformation A should be the most abundant conformer, the IPCM and SCRF values being ca. 100% and ca. 85% respectively. The IPCM calculations with the isodensity level set to 0.0005 present a conformational distribution close to that obtained from the hybrid model [92% (A) and 8% (B)], which has been considered our best solvent approach. Regarding the biological action of urea-type herbicides, the results presented here are important, because some QSAR studies have suggested that the partition coefficient is related to the herbicide activity, so the conformational equilibrium may play a role in the biological action. Received: 23 February 1998 / Accepted: 28 May 1998 / Published online: 19 August 1998     

Theoretical and experimental vibrational study of phenylurea: structure, solvent effect and inclusion process with the beta-cyclodextrin in the solid state

FTIR and Raman vibrational spectroscopic techniques as well as DFT quantum chemical calculation were used for characterizing conformational changes of phenylurea (a herbicide parent molecule) occurring from solid state to aqueous medium. Experimental infrared frequencies were assigned on the base of urea and benzenic derivatives spectroscopic data available in the literature and vibrational normal modes analytical calculation at the fully optimized geometry. Investigation of isotopic and solvent effects has revealed that the structure of phenylurea is particularly sensitive to the electrostatic environment via hydrogen non covalent bonds. The ability of beta-cyclodextrin (beta-CD) to form host-guest inclusion complex with phenylurea in the solid state was also evidenced by significant frequency shifts observed in the 1400-1800 cm(-1) spectral range.     

A molecular mechanics and semiempirical conformational analysis of the herbicide diuron inhibitor of photosystem II

The potential energy surface (PES) for the herbicide diuron (DCMU), a photosystem II inhibitor, has been extensively investigated using the quantum-mechanical semiempirical molecular orbital methods AM1 and PM3 and molecular mechanics method. A detailed conformational search has been carried out which revealed the occurrence of four genuine minimum energy structures. The relative stability of the conformers and rotational barriers to conformational interconversion were evaluated using distinct theoretical approaches. The results showed that thetrans form of the diuron molecule is more stable than thecis form in all methods, and so it may possibly be the biologically active isomer.     

Time-dependent conformational change of thrombin molecules induced by sulfated polysaccharides

Dextran sulfate (DS) had a greater ability to elute thrombin adsorbed on a small Sepharose 6B column than heparin did, while chondroitin sulfate A had little ability. It is probable that the strength of the interaction of thrombin depends mostly on the charge-density of strongly acidic sulfate groups in the polysaccharides. The change in intrinsic fluorescence intensity of thrombin with time was closely correlated with the rate of inactivation of the enzyme in the presence of sulfated polysaccharides. Both rates were affected by the pH of the solution in the presence of the polyanions. The rates in the presence of DS were highest at pH 6.05 among the three pHs tested, while they were enhanced only at pH 6.05 by heparin, but not by chondroitin sulfate A. Therefore, extensive charge-neutralization of thrombin by the sulfated polysaccharides is able to induce time- and temperature-dependent intramolecular conformational change (irreversible denaturation) of the enzyme molecules.     

Elastic properties of single amylose chains in water: a quantum mechanical and AFM study

Recent single-molecule atomic force microscopy (AFM) experiments have revealed that some polysaccharides display large deviations from force-extension relationships of other polymers which typically behave as simple entropic springs. However, the mechanism of these deviations has not been fully elucidated. Here we report the use of novel quantum mechanical methodologies, the divide-and-conquer linear scaling approach and the self-consistent charge density functional-based tight binding (SCC-DFTB) method, to unravel the mechanism of the extensibility of the polysaccharide amylose, which in water displays particularly large deviations from the simple entropic elasticity. We studied the deformations of maltose, a building block of amylose, both in a vacuum and in solution. To simulate the deformations in solution, the TIP3P molecular mechanical model is used to model the solvent water, and the SCC-DFTB method is used to model the solute. The interactions between the solute and water are treated by the combined quantum mechanical and molecular mechanical approach. We find that water significantly affects the mechanical properties of maltose. Furthermore, we performed two nanosecond-scale steered molecular dynamics simulations for single amylose chains composed of 10 glucopyranose rings in solution. Our SCC-DFTB/MM simulations reproduce the experimentally measured force-extension curve, and we find that the force-induced chair-to-boat transitions of glucopyranose rings are responsible for the characteristic plateau in the force-extension curve of amylose. In addition, we performed single-molecule AFM measurements on carboxymethyl amylose, and we found that, in contrast to the results of an earlier work by others, these side groups do not significantly affect amylose elasticity. By combining our experimental and modeling results, we conclude that the nonentropic elastic behavior of amylose is governed by the mechanics of pyranose rings themselves and their force-induced conformational transitions.     

Can a synthetic thread act as an electrochemically switchable molecular device?

Here we report that at room temperature in acetonitrile after the reduction of the naphthalimide-site, a synthetic molecular thread undergoes a complete conformational change which makes possible an efficient conversion of chemical energy into mechanical work; such results point out the ability of the thread to act as a molecular device under electrochemical control.     

Relaxation behavior,intramolecular interactions,and local motions on monosubstituted esters of poly(itaconic acid)

The mechanical relaxation spectrum of poly(monocyclohexylmethylene itaconate) (PMCMI) exhibits two well-developed absorptions in the glassy state that in increasing temperature order are named γ and β absorptions. Owing to the restricted conformational versatility of the backbone, the polymer presents a weak glass-rubber relaxation whose intensity is significantly lower than that of the γ absorption. Comparison of the mechanical spectrum of this polymer with that of poly(dicyclohexylmethylene itaconate) (PDCMI) allows the conclusion that the β relaxation is produced by motions in which the ? COOCH₂C₆H₁₁ side groups are involved. The location of the mechanical γ peak suggests that this absorption is produced by flipping conformational transitions in the cyclohexyl residue. Three dielectric absorptions are observed in the glassy state of PMCMI which in increasing temperature order are called δ, γ, and β relaxations. Both the location and the activation energy of the dielectric and mechanical β absorptions suggest that both relaxations are caused by the same molecular motions. Dipolar interactions in the liquid and glassy state are calculated and the results compared with those experimentally evaluated. © 1994 John Wiley & Sons, Inc.     

Functionality of photosynthetic reaction centers in polyelectrolyte multilayers: toward an herbicide biosensor

The bacterial reaction center (RC), a membrane photosynthetic protein, has been adsorbed onto a glass surface by alternating deposition with the cationic polymer poly(dimethyldiallylammonium chloride) (PDDA) obtaining as an end result an ordinate polyelectrolyte multilayer (PEM) where the protein retains its integrity and photoactivity over a period of several months. Such a system has been characterized from the functional point of view by checking the protein photoactivity at different hydration conditions, from extensive drought to full hydration. The kinetic analysis of charge recombination indicates that incorporation of RCs into dehydrated PEM hinders the conformational dynamics gating QA- to QB electron-transfer leaving unchanged the protein relaxation that stabilizes the primary charge separated state P+QA-. The herbicide-induced inhibition of the QB activity was studied in some detail. By dipping the PEM in herbicide solutions for short times, kinetics of herbicide binding and release have been determined; binding isotherms have been studied using PEM immersed in herbicide solution. QB functionality of RC has been restored by rinsing the PEM with water, thus allowing the reuse of the same sample. This last point has been exploited to design a simple optical biosensor for herbicides. A suitable kinetic model has been proposed to describe the interplay between forward and back electron-transfer processes upon continuous illumination, and the use of the PDDA-RC multilayers in herbicide bioassays was successfully tested.     

Probing Helical Hydrophobic Binding Sites in Branched Starch Polysaccharides Using NMR Spectroscopy

Branched starch polysaccharides are capable of binding multiple hydrophobic guests, but their exploitation as multivalent hosts and in functional materials is limited by their structural complexity and diversity. Linear α(1–4)‐linked glucose oligosaccharides are known to bind hydrophobic guests inside left‐handed single helices in solution and the solid state. Here, we describe the development of an amphiphilic probe that binds to linear α(1–4)‐linked glucose oligosaccharides and undergoes a conformational switch upon complexation, which gives rise to dramatic changes in the ¹H NMR spectrum of the probe. We use this probe to explore hydrophobic binding sites in the branched starch polysaccharides amylopectin and β‐limit dextrin. Diffusion‐ordered (DOSY), nuclear Overhauser effect (NOESY) and chemical shift perturbation (HSQC) NMR experiments are utilised to provide evidence that, in aqueous solution, branched polysaccharides bind hydrophobic guests in well‐defined helical binding sites, similar to those reported for complexation by linear oligosaccharides. By examining the binding affinity of the probe to systematically enzymatically degraded polysaccharides, we deduce that the binding sites for hydrophobic guests can be located on internal as well as external branches and that proximal α(1–6)‐linked branch points weaken but do not prevent complexation.     

Durable antifog films from layer-by-layer molecularly blended hydrophilic polysaccharides

Mechanically durable, long-lasting antifog coatings based on polysaccharides were developed using a layer-by-layer (LBL) assembly process. The unique properties of these coatings are a result of a molecular-level blending of the polysaccharides, with multilayers containing chitosan and carboxymethyl cellulose providing the best overall properties. The antifog properties resulted from a strong interaction between the polar and H-bonding elements of the assembled polymers and water molecules and the concomitant formation of thin films of water. Environmental scanning electron microscopy (ESEM) studies confirmed that fogging coatings are decorated with light scattering, micrometer-sized droplets of water whereas antifogging coatings remain droplet free. To improve the mechanical durability of the multilayer films on substrates, the surface was modified via self-assembly of epoxy-functionalized silane molecules. Cross-linking chemistry was then applied to improve the mechanical robustness of the LBL films on various surfaces. These films were characterized using several techniques: optical profilometery (PL), spectroscopic ellipsometry (EL), contact angle goniometry (CA), and atomic force microscopy (AFM). The antifog properties of the films were evaluated by several tests under different environmental conditions. This work demonstrates that the unique water-adsorbing properties of polysaccharides can be exploited to create permanent antifog properties, which may be useful for various applications.     

Acetate-methyl signals in the nuclear magnetic resonance spectra of peracetylated derivatives of some oligo- and polysaccharides

Peracetyl derivatives of oligo- and polysaccharides showed their own characteristic acetate-methyl signals in the NMR spectra measured in chloroform-d, which are useful for the identification and conformational analysis of oligo- and polysaccharides in solutions. An acetate-methyl signal in the lowest field among three acetate-methyl signals appeared in the NMR spectra of amylose acetate and was assigned to the acetate-methyl signal at C₆ on the basis of acetate-methyl signals of peracetylated derivatives of 6-O-tosylamylose and xylan. Monosaccharide moieties in oligo- and polysaccharides examined were found in the C1 (D ) conformation.     

The role of nanocellulose fibers, starch and chitosan on multipolysaccharide based films

Thin nanocomposite films of thermoplastic starch, chitosan and cellulose nanofibers (bacterial cellulose or nanofibrillated cellulose) were prepared for the first time by solvent casting of water based suspensions of the three polysaccharides. The role of the different bioploymers on the final properties (thermal stability, transparency, mechanical performance and antimicrobial activity) of the films was related with their intrinsic features, contents and synergic effects resulting from the establishment of interactions between them. Thermoplastic starch displays an important role on the thermal stability of the films because it is the most stable polysaccharide; however it has a negative impact on the mechanical performance and transparency of the films. The addition of chitosan improves considerably the transparency (up to 50 % transmittance for 50 % of chitosan, in respect to the amount of starch), mechanical performance and antimicrobial properties (at least 25 % of chitosan and no more than 10 % of cellulose nanofibers are required to observe bacteriostatic or bactericidal activity) but decrease their thermal stability. The incorporation of cellulose nanofibers had the strongest positive impact on the mechanical properties of the materials (increments of up to 15 and 30 MPa on the Young′s modulus and Tensile strength, respectively, for films with 20 % of BC or NFC). Nonetheless, the impact in thermal stability and mechanical performance of the films, promoted by the addition of chitosan and cellulose nanofibres, respectively, was higher than the expected considering their percentage contents certainly because of the establishment of strong and complex interactions between the three polysaccharides.     

Comprehensive review in moisture retention mechanism of polysaccharides from algae,plants, bacteria and fungus

Polysaccharides have attracted much attention due to their significant bio-activities. This review aims to summarize the main polysaccharides sources of related polysaccharides from algae, plants, fungus, and bacteria, and give insights into the structure–activity relationship between the moisture retention and structural characteristics of polysaccharides. The molecular weight, functional groups, polysaccharide modifications and apparent structure of polysaccharides are closely related to the moisturizing properties of polysaccharides in terms of moisturizing conformation. Based on recent moisturizing pieces of evidence, we propose a new framework focusing on the moisturizing intrinsic and extrinsic mechanisms. Polysaccharides molecular weight has different effects on moisturizing property. The extrinsic moisturization is mainly via the formation of hydrogen bonds between polysaccharides, the intrinsic moisturizing is mainly by regulating the production of some tight junction proteins. Accordingly, this review could further open the door for the production and application of polysaccharides as novel moisturizing agents in the cosmetic field.     

Synthesis and investigation of polyethylene blends with natural polysaccharides and their derivatives

Powder blends of LDPE with cellulose, ethyl cellulose, starch, chitin, and chitosan have been prepared under shear deformation in a rotor disperser at different initial-component ratios. The composition of powder fractions is identical to the original composition of the blends. The studied polymer blends demonstrate high mechanical characteristics. X-ray diffraction analysis and DSC studies show that the blending of LDPE with polysaccharides under shear deformations results in changes in the polymer structure and leads to a decrease of their degree of crystallinity. The maximum intensity of mold fungi growth is observed in starch-LDPE (50: 50, wt/wt) and chitin-LDPE (50: 50, wt/wt) blends.     

Stretching single polysaccharides and proteins using atomic force microscopy

The past years have witnessed remarkable advances in our use of atomic force microscopy (AFM) for stretching single biomolecules, thereby contributing to answering many outstanding questions in biophysics and chemical biology. In these single-molecule force spectroscopy (SMFS) experiments, the AFM tip is continuously approached to and retracted from the biological sample, while monitoring the interaction force. The obtained force-extension curves provide key insight into the molecular elasticity and localization of single molecules, either on isolated systems or on cellular surfaces. In this tutorial review, we describe the principle of such SMFS experiments, and we survey remarkable breakthroughs made in manipulating single polysaccharides and proteins, including understanding the conformational properties of sugars and controlling them by force, measuring the molecular elasticity of mechanical proteins, unfolding and refolding individual proteins, probing protein-ligand interactions, and tuning enzymatic reactions by force. In addition, we show how SMFS with AFM tips bearing specific bioligands has enabled researchers to stretch and localize single molecules on live cells, in relation with cellular functions.     

Effect of chain aggregation and ionic interactions on the proton dissociation equilibria of weak polyacids

A theoretical interpretation of the dissociation behavior of weak polyacids is given. Incorporation of conformational variability in the free energy of a polyelectrolyte system extends the validity of the counterion condensation theory to cases in which cooperative transitions and/or aggregation phenomena take place. Comparison of the theoretical predictions with experimental data on ionic polysaccharides, either natural or derivatives, is presented.     

Free energy surfaces for the alpha(1 --> 4)-glycosidic linkage: implications for polysaccharide solution structure and dynamics

We present a potential of mean force surface for rotation about phi and psi dihedral angles of the alpha(1 --> 4)-glycosidic linkage in the maltose disaccharide (4-O-alpha-d-glucopyranosyl-d-glucopyranose) in aqueous solution. Comparison of the vacuum and solution free energy surfaces for maltose shows the principal effects of water to be an increase in the rotational freedom of the alpha(1 --> 4) linkage brought about by lowering the energy barrier for syn to anti conformational changes as well as expansion of the range of low-energy phi,psi conformations. This free energy analysis thus provides a thermodynamic and conformational rationale for the effects of water on alpha(1 --> 4)-linked polysaccharides and carbohydrate glasses.