Correlation Between Chain Architecture and Hydration Water Structure in Polysaccharides

The physical properties of confined water can differ dramatically from those of bulk water. Hydration water associated with polysaccharides provides a particularly interesting example of confined water, because differences in polysaccharide structure provide different spatially confined environments for water sorption. We have used attenuated total reflection infrared (ATR-IR) spectroscopy to investigate the structure of hydration water in films of three different polysaccharides under controlled relative humidity (RH) conditions. We compare the results obtained for films of highly branched, dendrimer-like phytoglycogen nanoparticles to those obtained for two unbranched polysaccharides, hyaluronic acid (HA), and chitosan. We find similarities between the water structuring in the two linear polysaccharides and significant differences for phytoglycogen. In particular, the results suggest that the high degree of branching in phytoglycogen leads to a much more well-ordered water structure (low density, high connectivity network water), indicating the strong influence of chain architecture on the structuring of water. These measurements provide unique insight into the relationship between the structure and hydration of polysaccharides, which is important for understanding and exploiting these sustainable nanomaterials in a wide range of applications

Using Nanoscale Substrate Curvature to Control the Dimerization of a Surface-Bound Protein

The influence of surface geometry on adsorbed proteins offers new possibilities for controlling quaternary structure by manipulating protein–protein interactions at a surface, with applications that are relevant to protein aggregation, fibrillation, ligand binding, and surface catalysis. To understand the effect of surface curvature on the structure of the surface-bound protein β-lactoglobulin (β-LG), we have used a combination of polystyrene (PS) nanoparticles (NPs) and ultrathin PS films to fabricate chemically pure, hydrophobic surfaces that have nanoscale curvature and are stable in aqueous buffer. We have used single molecule force spectroscopy to measure the detachment contour lengths <i>L</i>_c for β-LG adsorbed on the highly curved PS surfaces, and we compare these values <i>in situ</i> to those measured for β-LG adsorbed on flat PS surfaces on the same samples. The <i>L</i>_c distributions measured on all flat PS surfaces show a large monomer peak near 60 nm and a smaller dimer peak at 120 nm. For 190 and 100 nm diameter NPs, which are effectively flat on the scale of the β-LG molecules, there is no measurable difference between the <i>L</i>_c distributions obtained for the flat and curved surfaces. However, for 60 nm diameter NPs the dimer peak is smaller, and for 25 nm diameter NPs the dimer peak is absent, indicating that the number of surface-bound dimers is significantly reduced by an increase in the curvature of the underlying surface. These results indicate that surface curvature provides a new method of manipulating protein–protein interactions and controlling the quaternary structure of adsorbed proteins

Efficient Modeling of High-Generation Dendrimers in Solution Using Dynamical Self-Consistent Field Theory

We extend dynamical self-consistent field theory (dSCFT) to large, nonlinear polymer chains to simulate the evolution of high-generation dendrimers in a solvent. Because the number of beads N within these bead–spring dendrimers is very large, we introduce a numerical technique to efficiently analyze the Rouse modes of the dendrimer through a decomposition of the dendrimer into many smaller subchains, achieving a significant improvement, from O(N2) to O(N), in the scaling of the simulation time for the Rouse motion of the dendrimer. By adjusting the strength of the interaction between dendrimer and solvent beads, we obtain qualitative and quantitative agreement with the core-chain morphology, 22 nm radius, and high degree of hydration measured experimentally using small-angle neutron scattering for 11-generation, glucose-based phytoglycogen dendrimers in water, validating dSCFT in this context

Equilibrium Swelling, Interstitial Forces, and Water Structuring in Phytoglycogen Nanoparticle Films

Phytoglycogen is a highly branched polymer of glucose that forms dendrimeric nanoparticles. This special structure leads to a strong interaction with water that produces exceptional properties such as high water retention, low viscosity, and high stability of aqueous dispersions. We have used ellipsometry at controlled relative humidity (RH) to measure the equilibrium swelling of ultrathin films of phytoglycogen, which directly probes the interstitial forces acting within the films. Comparison of the swelling behavior of films of highly branched phytoglycogen to that of other glucose-based polysaccharides shows that the chain architecture plays an important role in determining both the strong, short-range repulsion of the chains at low RH and the repulsive hydration forces at high RH. In particular, the length scale λ₀ that characterizes the exponentially decaying hydration forces provides a quantitative, RH-independent measure of film swelling that differs significantly for different glucose-based polysaccharides. By combining ellipsometry with infrared spectroscopy, we have determined the relationship between water structuring and inter-chain separation in the highly branched phytoglycogen nanoparticles, with maintenance of a high degree of water structure as the film swells significantly at high RH. These insights into the structure–hydration relationship for phytoglycogen are essential to the development of new products and technologies based on this sustainable nanomaterial

Direct in Situ Observation of Synergism between Cellulolytic Enzymes during the Biodegradation of Crystalline Cellulose Fibers

High-resolution atomic force microscopy (AFM) was used to image the real-time in situ degradation of crystalline by three types of <i>T. reesei</i> cellulolytic enzymesTrCel6A, TrCel7A, and TrCel7Band their mixtures. TrCel6A and TrCel7A are exo-acting cellobiohydrolases processing cellulose fibers from the nonreducing and reducing ends, respectively. TrCel7B is an endoglucanase that hydrolyzes amorphous cellulose within fibers. When acting alone on native cellulose fibers, each of the three enzymes is incapable of significant degradation. However, mixtures of two enzymes exhibited synergistic effects. The degradation effects of this synergism depended on the order in which the enzymes were added. Faster hydrolysis rates were observed when TrCel7A (exo) was added to fibers pretreated first with TrCel7B (endo) than when adding the enzymes in the opposite order. Endo<i>-</i>acting TrCel7B removed amorphous cellulose, softened and swelled the fibers, and exposed single microfibrils, facilitating the attack by the exo-acting enzymes. AFM images revealed that exo-acting enzymes processed the TrCel7B-pretreated fibers preferentially from one specific end (reducing or nonreducing). The most efficient (almost 100%) hydrolysis was observed with the mixture of the three enzymes. In this mixture, TrCel7B softened the fiber and TrCel6A and TrCel7A were directly observed to process it from the two opposing ends. This study provides high-resolution direct visualization of the nature of the synergistic relation between <i>T. reesei </i>exo- and endo-acting enzymes digesting native crystalline cellulose

Direct in Situ Observation of Synergism between Cellulolytic Enzymes during the Biodegradation of Crystalline Cellulose Fibers

High-resolution atomic force microscopy (AFM) was used to image the real-time in situ degradation of crystalline by three types of <i>T. reesei</i> cellulolytic enzymesTrCel6A, TrCel7A, and TrCel7Band their mixtures. TrCel6A and TrCel7A are exo-acting cellobiohydrolases processing cellulose fibers from the nonreducing and reducing ends, respectively. TrCel7B is an endoglucanase that hydrolyzes amorphous cellulose within fibers. When acting alone on native cellulose fibers, each of the three enzymes is incapable of significant degradation. However, mixtures of two enzymes exhibited synergistic effects. The degradation effects of this synergism depended on the order in which the enzymes were added. Faster hydrolysis rates were observed when TrCel7A (exo) was added to fibers pretreated first with TrCel7B (endo) than when adding the enzymes in the opposite order. Endo<i>-</i>acting TrCel7B removed amorphous cellulose, softened and swelled the fibers, and exposed single microfibrils, facilitating the attack by the exo-acting enzymes. AFM images revealed that exo-acting enzymes processed the TrCel7B-pretreated fibers preferentially from one specific end (reducing or nonreducing). The most efficient (almost 100%) hydrolysis was observed with the mixture of the three enzymes. In this mixture, TrCel7B softened the fiber and TrCel6A and TrCel7A were directly observed to process it from the two opposing ends. This study provides high-resolution direct visualization of the nature of the synergistic relation between <i>T. reesei </i>exo- and endo-acting enzymes digesting native crystalline cellulose

Direct in Situ Observation of Synergism between Cellulolytic Enzymes during the Biodegradation of Crystalline Cellulose Fibers

High-resolution atomic force microscopy (AFM) was used to image the real-time in situ degradation of crystalline by three types of <i>T. reesei</i> cellulolytic enzymesTrCel6A, TrCel7A, and TrCel7Band their mixtures. TrCel6A and TrCel7A are exo-acting cellobiohydrolases processing cellulose fibers from the nonreducing and reducing ends, respectively. TrCel7B is an endoglucanase that hydrolyzes amorphous cellulose within fibers. When acting alone on native cellulose fibers, each of the three enzymes is incapable of significant degradation. However, mixtures of two enzymes exhibited synergistic effects. The degradation effects of this synergism depended on the order in which the enzymes were added. Faster hydrolysis rates were observed when TrCel7A (exo) was added to fibers pretreated first with TrCel7B (endo) than when adding the enzymes in the opposite order. Endo<i>-</i>acting TrCel7B removed amorphous cellulose, softened and swelled the fibers, and exposed single microfibrils, facilitating the attack by the exo-acting enzymes. AFM images revealed that exo-acting enzymes processed the TrCel7B-pretreated fibers preferentially from one specific end (reducing or nonreducing). The most efficient (almost 100%) hydrolysis was observed with the mixture of the three enzymes. In this mixture, TrCel7B softened the fiber and TrCel6A and TrCel7A were directly observed to process it from the two opposing ends. This study provides high-resolution direct visualization of the nature of the synergistic relation between <i>T. reesei </i>exo- and endo-acting enzymes digesting native crystalline cellulose

Infrared Studies of the Potential Controlled Adsorption of Sodium Dodecyl Sulfate at the Au(111) Electrode Surface

Quantitative subtractively normalized interfacial Fourier transform infrared reflection spectroscopy (SNIFTIRS) was used to determine the conformation and orientation of sodium dodecyl sulfate (SDS) molecules adsorbed at the single crystal Au(111) surface. The SDS molecules form a hemimicellar/hemicylindrical (phase I) structure for the range of potentials between −200 ≤ <i>E</i> < 450 mV and condensed (phase II) film for electrode potentials ≥500 mV vs Ag/AgCl. The SNIFTIRS measurements indicate that the alkyl chains within the two adsorbed states of SDS film are in the liquid-crystalline state rather than the gel state. However, the sulfate headgroup is in an oriented state in phase I and is disordered in phase II. The newly acquired SNIFTIR spectroscopy measurements were coupled with previous electrochemical, atomic force microscopy, and neutron reflectivity data to improve the current existing models of the SDS film adsorbed on the Au(111) surface. The IR data support the existence of a hemicylindrical film for SDS molecules adsorbed at the Au(111) surface in phase I and suggest that the structure of the condensed film in phase II can be more accurately modeled by a disordered bilayer

Electrochemical and PM-IRRAS Characterization of Cholera Toxin Binding at a Model Biological Membrane

A mixed phospholipid-cholestrol bilayer, with cholera toxin B (CTB) units attached to the monosialotetrahexosylganglioside (GM1) binding sites in the distal leaflet, was deposited on a Au(111) electrode surface. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were used to characterize structural and orientational changes in this model biological membrane upon binding CTB and the application of the electrode potential. The data presented in this article show that binding cholera toxin to the membrane leads to an overall increase in the tilt angle of the fatty acid chains; however, the conformation of the bilayer remains relatively constant as indicated by the small decrease in the total number of gauche conformers of acyl tails. In addition, the bound toxin caused a significant decrease in the hydration of the ester group contained within the lipid bilayer. Furthermore, changes in the applied potential had a minimal effect on the overall structure of the membrane. In contrast, our results showed significant voltage-dependent changes in the average orientation of the protein α-helices that may correspond to the voltage-gated opening and closing of the central pore that resides within the B subunit of cholera toxin

Real-Time Observation of the Swelling and Hydrolysis of a Single Crystalline Cellulose Fiber Catalyzed by Cellulase 7B from <i>Trichoderma reesei</i>

The biodegradation of cellulose involves the enzymatic action of cellulases (endoglucanases), cellobiohydrolases (exoglucanases), and β-glucosidases that act synergistically. The rate and efficiency of enzymatic hydrolysis of crystalline cellulose in vitro decline markedly with time, limiting the large-scale, cost-effective production of cellulosic biofuels. Several factors have been suggested to contribute to this phenomenon, but there is considerable disagreement regarding the relative importance of each. These earlier investigations were hampered by the inability to observe the disruption of crystalline cellulose and its subsequent hydrolysis directly. Here, we show the application of high-resolution atomic force microscopy to observe the swelling of a single crystalline cellulose fiber and its-hydrolysis in real time directly as catalyzed by a single cellulase, the industrially important cellulase 7B from <i>Trichoderma reesei</i>. Volume changes, the root-mean-square roughness, and rates of hydrolysis of the surfaces of single fibers were determined directly from the images acquired over time. Hydrolysis dominated the early stage of the experiment, and swelling dominated the later stage. The high-resolution images revealed that the combined action of initial hydrolysis followed by swelling exposed individual microfibrils and bundles of microfibrils, resulting in the loosening of the fiber structure and the exposure of microfibrils at the fiber surface. Both the hydrolysis and swelling were catalyzed by the native cellulase; under the same conditions, its isolated carbohydrate-binding module did not cause changes to crystalline cellulose. We anticipate that the application of our AFM-based analysis on other cellulolytic enzymes, alone and in combination, will provide significant insight into the process of cellulose biodegradation and greatly facilitate its application for the efficient and economical production of cellulosic ethanol