An NMR relaxation and spin diffusion study of cellulose structure during water adsorption

The goal of this paper is a systematic investigation of changes in the supramolecular structure of cellulose during its water uptake. The main attention is concentrated on the analysis of the mechanism of dispersion of microfibrils by proton NMR relaxation techniques. Spin diffusion NMR experiments made it possible to estimate the linear dimensions of the surface thickness of cellulose crystallites and the average depth of micropores that are formed between elementary fibrils, as well as the character of the filling of micropores during adsorption. It has been shown that when the relative water content gradually increases to 7–8%, water molecules occupy the space between cellulose microfibrils, which is accompanied by an increase in the pore sizes and their specific surface area and a simultaneous decrease in the degree of crystallinity. Upon acquiring a free induction decay signal, a magic sandwich echo sequence was used, due to which the accuracy and information value of the results were considerably improved.     

NMR study on molecular mobility of DNA molecules in solution

The molecular mobility of calf thymus DNA molecules in solution has been discussed in terms of correlation time τ calculated from measurements of longitudinal T₁ and transverse T₂ magnetic relaxation times. The influence of DNA concentration and ionic strength of the solution upon freedom of movement of DNA molecules was studied for native and denatured DNA and also during thermal helix-coil transition. The dependence of τ values on temperature was carried out by comparing the values of correlation times τ_tat given temperature with the correlation time τ₂₀ at 20°C. The molecular rotation of DNA at 20°C and at higher ionic strength at 0.15 and 1.0.M NaCl is described by τ values of the order of 1.0–1.2 × 10^?8 and was reduced slightly with increase of temperature below the helix-coil transition. The molecular rotation of DNA in 0.02MNaCl was lower at 20°C as compared to DNA in solvents with higher NaCl concentrations and increases rapidly with increase of temperature in the range 20–60°C. The values of correlation time are characterized by fast increase at temperatures above the spectrophotometrically determined beginning of melting curve. The beginning of this increase is observed at about 65, 80, and 85°C for DNA in 0.02, 0.15, and 1.0MNaCl, respectively. Values of correlation time for denatured DNA are in all cases about 1.1–1.4 times that for native DNA. The obtained results are discussed in terms of conformation of DNA molecules in solution as well as in terms of water dipole binding in DNA hydration shells.     

Preservation of isoamylase adsorbed onto raw corn starch

Isoamylase produced by Pseudomonas amyloderamosa was adsorbed to near-completion on to raw corn starch. The isoamylase/starch granules when freeze-dried were stable at 4–6°C, but not at room temperature, for 8 months. Granules prepared by vacuum-drying method were stable at both temperatures for the same time.     

Transient amidolytic activity changes of plasmin adsorbed onto carbons

The amidolytic activity of plasmin (Pln) that spontaneously adsorbs from solutions to modified-graphite (GR) and glassy carbon (GL) surfaces was studied in the 10-45 degrees C temperature range in the presence of a chromogenic substrate. Surfaces were modified with a coating of fibrinogen, either electrochemically oxidized or not. The effect of an additional modification via deposition of Langmuir-Blodgett films of behenic acid (BA-LB) onto the former surfaces, thus leading to either hydrophobic or hydrophilic surfaces according to the number of deposed layers, was examined. In all cases results showed the occurrence of a first order transition which strongly and transiently increases the surface activity of Pln. At BA-LB surfaces, the most prominent change compared with other coatings was a significant enhancement of the critical temperature Tc that characterizes the beginning of the transition. When fibrinogen was present in the solution, the transition was no longer observable up to 37 degrees C as shown by the linear kinetics exhibited by surfaces bearing oxidized fibrinogen.     

NMR study of cellulose and wheat straw degradation by Ruminococcus albus 20

Cellulose and wheat straw degradation by Ruminococcus albus was monitored using NMR spectroscopy. In situ solid-state (13)C-cross-polarization magic angle spinning NMR was used to monitor the modification of the composition and structure of cellulose and (13)C-enriched wheat straw during the growth of the bacterium on these substrates. In cellulose, amorphous regions were not preferentially degraded relative to crystalline areas by R. albus. Cellulose and hemicelluloses were also degraded at the same rate in wheat straw. Liquid state two-dimensional NMR experiments were used to analyse in detail the sugars released in the culture medium, and the integration of NMR signals enabled their quantification at various times of culture. The results showed glucose and cellodextrin accumulation in the medium of cellulose cultures; the cellodextrins were mainly cellotriose and accumulated to up to 2 mm after 4 days. In the wheat straw cultures, xylose was the main soluble sugar detected (1.4 mm); arabinose and glucose were also found, together with some oligosaccharides liberated from hemicellulose hydrolysis, but to a much lesser extent. No cellodextrins were detected. The results indicate that this strain of R. albus is unable to use glucose, xylose and arabinose for growth, but utilizes efficiently xylooligosaccharides. R. albus 20 appears to be less efficient than Fibrobacter succinogenes S85 for the degradation of wheat straw.     

Proteomic analysis of bone proteins adsorbed onto the surface of titanium dioxide

Osseointegration is the structural and functional connection between bone tissues and implants such as titanium dioxide (TiO₂). The bone-TiO₂ interface is thought to contain proteoglycans. However, exhaustive analysis of the proteins in this layer has not been performed. In this study, we evaluated the bone protein adhered on the surface of TiO₂ comprehensively. Pig bone protein was extracted by sequential elutions with guanidine, 0.1 M EDTA, and again with guanidine. The proteins obtained from these extractions were allowed to adhere to an HPLC column packed with TiO₂ and were eluted with 0.2 M NaOH. The eluted proteins were identified by LC/MS/MS and included not only proteoglycans but also other proteins such as extracellular matrix proteins, enzymes, and growth factors. Calcium depositions were observed on TiO₂ particles incubated with bone proteins, guanidine-extracted proteins adhered to TiO₂ displayed significantly high amounts of calcium depositions.     

Compaction of single supercoiled DNA molecules adsorbed onto amino mica

A model of possible conformational transitions of supercoiled DNA in vitro in the absence of proteins under the conditions of increasing degree of compaction was developed. A 3993-bp pGEMEX supercoiled DNA immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica with a hydrophobicity higher than that of standard amino mica) was visualized by atomic force microscopy in air. On the modified amino mica, which has an increased density of surface positive charges, single molecules with an extremely high degree of compaction were visualized in addition to plectonemic DNA molecules. As the degree of DNA supercoiling increased, the length of the first-order superhelical axis of molecules decreased from 570 to 370 nm, followed by the formation of second- and third-order superhelical axes about 280 and 140 nm long, respectively. The compaction of molecules ends with the formation of minitoroids about 50 nm in diameter and molecules of spherical shape. It was shown that the compaction of single supercoiled DNA molecules immobilized on amino mica to the level of minitoroids and spheroids is due to the shielding of mutually repulsing negatively charged phosphate groups of DNA by positively charged amino groups of the amino mica, which has a high charge density of its surface.     

Oxygen-17 NMR relaxation studies on gelatinization temperature and water mobility in maize starches

The effects of starch/water ratio, amylose content, degree of phosphorylation, and added KI on water mobility in maize starch-water dispersions were studied by oxygen-17 spin-spin relaxation time measurements over a range of temperatures. The results demonstrate that: (i) the changes in spin-spin relaxation time (ΔT₂) reflect the degree of starch-water interaction at different stages of the heating process; (ii) the amount of added water affects the initial T₂ and ΔT₂ during gelatinization; (iii) higher amylose contents result in lower water mobility in starch-water systems; (iv) higher degrees of phosphorylation lead to a decrease in water mobility, accompanied by a decrease in gelatinization temperature; and (v) added KI effectively decreases water mobility and gelatinization temperature in the starch-water systems studied.     

Compaction of single supercoiled DNA molecules adsorbed onto amino mica

A model of possible conformational transitions of supercoiled DNA in vitro in the absence of proteins under the conditions of increasing degree of compaction was developed. A 3993-bp pGEMEX supercoiled DNA immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica with a hydrophobicity higher than that of standard amino mica) was visualized by atomic force microscopy in air. On the modified amino mica, which has an increased density of surface positive charges, single molecules with an extremely high degree of compaction were visualized in addition to plectonemic DNA molecules. As the degree of DNA supercoiling increased, the length of the first-order superhelical axis of molecules decreased from 570 to 370 nm, followed by the formation of second-and third-order superhelical axes about 280 and 140 nm long, respectively. The compaction of molecules ends with the formation of minitoroids about 50 nm in diameter and molecules of spherical shape. It was shown that the compaction of single supercoiled DNA molecules immobilized on amino mica to the level of minitoroids and spheroids is due to the shielding of mutually repulsing negatively charged phosphate groups of DNA by positively charged amino groups of the amino mica, which has a high charge density of its surface.     

Investigation of the state and dynamics of water in hydrogels of cellulose ethers by 1H NMR spectroscopy

The aim of this work was to study the effect of the type of substituent of the cellulose ethers and the molecular mass on the state and dynamics of water in the respective hydrogels to specify the quantity of adsorbed water on the polymers or, more explicitly, to calculate the average number of water molecules bound to a polymer repeating unit (PRU).¹H NMR relaxation experiments were performed on equilibrated systems of cellulose ether polymers (HEC, HPC, HPMC K4M, and HPMC K100M) with water. In particular, the water proton spinlattice (T ₁) and spin-spin (T ₂) relaxation times were measured in these systems at room temperature. The observed proton NMRT ₁ andT ₂ of water in hydrogels at different cellulose ether concentrations at room temperature were shown to decrease with increasing polymer concentration. The relaxation rate 1/T ₁ is sensitive to the type of polymer substituent but insensitive to the polymer molecular mass. The rate 1/T ₂ appears much less influenced by the polymer substitution. The procedure developed for calculating the amount of water bound per PRU, based on the analysis of theT ₁ andT ₂ data, shows that this amount is the largest for HPC followed by HEC, HP MC K4M, and HPMC K100M. The results correlate well with the degree of hydrophilic substitution of the polymer chains. This NMR analysis deals with a single molecular layer of adsorbed water for the investigated cellulose ether polymers at all concentrations, while the rest of the water in the hydrogel is bulk-like. Therefore, the mesh size of polymer network in the view of a single molecular layer is not effectively changed.     

NMR studies of mobility within protein structure

NMR studies of dynamics within structure have revealed that a quite new approach to protein structure and its relation to function is necessary. This approach requires the consideration in detail of the following: 1. Local movements of groups and small segments to allow fast recognition and fitting. The motion concerns on/off rates as well as binding. The observations affect surface/surface recognition, e.g. of antigen/antibody as well as of substrate and protein. 2. Somewhat larger interdomain or N- and C-terminal segments which allow rearrangement. Cases in point are the movement of segments in blood-clotting proteins or in histones. 3. Relative motion of helices in hinges. These actions are likely in such enzymes as kinases and P-450 cytochromes. 4. Relative motion of helices within domains (relative to other helices or sheets) in mechanical devices (triggers) e.g. in calmodulin. 5. General motion in random proteins. Examples extend from rubber-like proteins (entropy sensors), some glycoproteins, to proteins carrying peptide hormones to be generated only after hydrolysis. 6. Order----disorder transitions locally as in osteocalcin and metallothionine. 7. Swinging arm motions associated with special sequences such as (Ala-Pro)n. 8. Of great interest is the power of NMR to look at proteins which are relatively large, up to 50 kDa proteins, and to isolate certain zones of interest. This needs careful temperature dependent studies and analysis of separated domains [72] as well as the use of a great variety of pulse sequences [15] and of nuclei other than protons. 9. In this article I have illustrated the different possibilities using work in my own group. This is done to lessen the burden of extensive review. I fully realise that the range of examples is now large. I would stress though that the production of the necessary technology was the endeavour of several of us within the Oxford Enzyme Group from 1970 to 1985, i.e. from 270-600 MHz Fourier-transform NMR spectroscopy. 10. While all of these features have been demonstrated by NMR methods there are parallel developments both using X-ray diffraction methods and theoretical approaches. All these procedures are changing the view of protein structure to one which incorporates dynamics all the way from conventional vibronic/rotational coupling to the disordered motions characteristic of random polymers. It is the understanding of dynamics that leads to an appreciation of function.     

NMR spin-echo study of the effect of hydration on the mobility and conformation of linear polyethyleneimine]

It has been shown that mobility of protons in different groups of linear polyethylenimine (LPEI) is sensitive to the formation of H-bonds between amino groups of a polymer. Upon hydration of LPEI most of direct bonds between amino groups are substituted by the bonds via water molecules. This results in a conformational change of the polymer. Similar process may also occur in biopolymers.     

MD simulation of a plastocyanin mutant adsorbed onto a gold surface

MD simulation of plastocyanin, an electron transfer protein, adsorbed onto a gold surface, has been performed for 10 ns. Starting from the crystallographic structure of a poplar plastocyanin mutant engineered with the insertion of a disulfide bridge, the protein has been anchored to a gold substrate modeled by a cluster of three layers in the Au<111> configuration. A number of significant structural and dynamical properties of the protein molecule, covalently bound through either one or two sulfur atoms to the gold surface, has been extracted and compared with those of the free protein. Attention has been paid to investigate the dynamical aspects putatively related to the electron transfer process. In particular, the cross-correlation function between specific active site vibrations and all the other protein atom motions and the principal component analysis have been calculated in order to put into evidence dynamical correlation of some functional relevance. The results are discussed also in connection with related experiments.     

Secondary structure of proteins adsorbed onto or embedded in polyelectrolyte multilayers

The structural changes of bovine serum albumin (BSA) and hen egg white lysozyme (HEL) upon their adsorption onto the surface or their embedding into the interior of poly(allylamine hydrochloride)-(poly(styrenesulfonate) (PAH-PSS) multilayer architectures were investigated by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The presence of the polyelectrolytes seems, as previously observed for fibrinogen (J. Phys. Chem. B 2001, 105, 11906-11916), to prevent intermolecular interactions and, thus, protein aggregation at ambient temperature. The secondary structure of the proteins was somewhat altered upon adsorption onto the polyelectrolyte multilayers. The structural changes were larger when the charges of the multilayer outer layer and the protein were opposing. The adsorption of further polyelectrolyte layers onto protein-terminated architectures (i.e., embedding the proteins into a polyelectrolyte multilayer) did not cause considerable further changes in their secondary structures. The capacity of the polyelectrolyte architectures to delay the formation of intermolecular beta-sheets upon increasing temperatures was not uniform for the studied proteins. PSS in contact with HEL could largely prevent the heat-induced aggregation of HEL. In contrast, PAH had hardly any effect on the aggregation of BSA. The differences are explained on the basis of protein-polyelectrolyte interactions, affected mostly by the nature and the strength of the ionic interactions between the polyelectrolyte-protein contact surfaces.     

Mechanisms of increased immunogenicity for DNA-based vaccines adsorbed onto cationic microparticles

Investigation into the mechanism of action of vaccine adjuvants provides opportunities to define basic immune principles underlying the induction of strong immune responses and insights useful for the rational development of subunit vaccines. A novel HIV vaccine composed of plasmid DNA-encoding p55 gag formulated with poly-lactide-co-glycolide microparticles (PLG) and cetyl trimethyl ammonium bromide (CTAB) elicits both serum antibody titers and cytotoxic lymphocyte activity in mice at doses two orders of magnitude lower than those required for comparable response to plasmid DNA in saline. Using this model, we demonstrated the increase in potency requires the DNA to be complexed to the PLG-CTAB microparticles. Furthermore, the PLG-CTAB-DNA formulation increased the persistence of DNA at the injection site, recruited mononuclear phagocytes to the site of injection, and activated a population of antigen presenting cells. Intramuscular immunization with the PLG-CTAB-DNA complex induced antigen expression at both the injection site and the draining lymph node. These findings demonstrate that the PLG-CTAB-DNA formulation exhibits multiple mechanisms of immunopotentiation.     

An NMR study of the conformational mobility of 7alpha,8alpha analogues of steroid estrogens

All the signals in the 1H and 13C NMR spectra of some analogues of 7alpha-methyl-8alpha- and 6-oxa-8alpha-steroid estrogens were completely assigned. Considering the values of nuclear Overhauser effect and vicinal coupling constants, these steroids were shown to exhibit a fast, on the NMR time scale, conformational equilibrium arising due to the inversion of ring B. The conformer populations were obtained from a comparison of the experimental and theoretical values of the dihedral angles and the interproton distances. This conformational equilibrium was shown to depend on the nature of atom in position 6: for the 7alpha-methyl-6-oxa-8alpha analogues of the steroid estrogens, the population of the conformer with the pseudoaxial orientation of the 7alpha-methyl group was observed to be decreased compared with the 7alpha-methyl-8alpha analogue.     

Kinetic modeling of cellulose hydrolysis with first order inactivation of adsorbed cellulase

Enzymatic hydrolysis involves complex interaction between enzyme, substrate, and the reaction environment, and the complete mechanism is still unknown. Further, glucose release slows significantly as the reaction proceeds. A model based on Langmuir binding kinetics that incorporates inactivation of adsorbed cellulase was developed that predicts product formation within 10% of experimental results for two substrates. A key premise of the model, with experimental validation, suggests that V(max) decreases as a function of time due to loss of total available enzyme as adsorbed cellulases become inactivated. Rate constants for product formation and enzyme inactivation were comparable to values reported elsewhere. A value of k(2)/K(m) that is several orders of magnitude lower than the rate constant for the diffusion-controlled encounter of enzyme and substrate, along with similar parameter values between substrates, implies a common but undefined rate-limiting step associated with loss of enzyme activity likely exists in the pathway of cellulose hydrolysis.