The reinforcement of gellan gel network by the immersion into salt solution

The effect of immersion into salt solutions on rheological properties of gellan gels was investigated. The storage Young's modulus of gellan gels increased with time during the immersion into salt solutions. The increase of the storage Young's modulus can not be explained solely by change in the concentration of gellan. The ellipticity at 202 nm decreased by the immersion, suggesting the formation and aggregation of gellan helices. It was considered that during immersion cations penetrated into gellan gels to induce the formation and aggregation of gellan helices in gels, resulting in reinforcement of the gel network.     

Binding effect of Cu(2+) as a trigger on the sol-to-gel and the coil-to-helix transition processes of polysaccharide, gellan gum

The binding effect of divalent cation Cu(2+) on the gelation process with a coil-helix transition in Cu(2+)/gellan aqueous solutions has been successfully elucidated by EPR, CD, and viscoelasticity measurements. Generally, Na-type gellan gum in aqueous solution can make gel when accompanied by an intrinsic coil-helix formation induced by hydrogen bonding between chains without any additional cations at T(ch)(-)(in) ( approximately 29 degrees C) with cooling temperature. An extrinsic coil-helix transition, induced by additional divalent cations in advance of the intrinsic sol-gel transition of gellan gum, is separately detected by CD measurement. The extrinsic coil-helix transition temperatures T(ch)(-)(ex) (>47 degrees C), which increased with the Cu(2+) concentration added, were nearly identical to the sol-gel transition temperature, T(sg), determined by the viscoelasticity measurement. Judging from the molar ellipticity by CD measurement and quantitative analysis of EPR spectra, it was elucidated that the helix forming process via divalent cations is composed of two steps ascribed to the different origins, i.e., a chemical binding effect via Cu(2+) ions in the initial stage and hydrogen bonds subsequently. Finally, we propose the coil-helix and the sol-gel transition mechanism initiated by the binding effect with the divalent cation, in which the partial chelate formation can cause local formation of helices and junction zones in the vicinity of the chelates at the initial stage of the process and stabilize the helices and the junction zones. On the other hand, the stabilized helices and junction zones can induce further formation and further stabilization of the Cu(2+)-gellan chelates. The mutual stabilization promotes the formation of three-dimensional network structure at the higher temperature than the intrinsic temperature for network formation.     

Light scattering studies of tetramethyl ammonium gellan

Tetramethyl ammonium (TMA) gellan does not gel. Light scattering studies suggest that in solutions of TMA gellan, in tetramethyl ammonium chloride (TMACI), the gellan molecules assemble end to end to produce elongated fibrous structures. Such fibrils are envisaged as resulting from double-helix formation between the ends of neighbouring gellan molecules. Fibrils with molecular weights ranging from (1.06 ± 0.06) × 10⁵ to (4.5 ± 0.1) × 10⁶ have been observed. The molecular weights obtained depended upon the pore size of the filters used to clarify the solutions. The formation of strong gels, in the presence of gel promoting cations, is attributed to a localized ordered lateral association, or crystallization of regions of these fibrils. It is suggested that such a model for gelation may be of general applicability to a number of polysaccharide systems.     

X-ray and computer modeling studies on gellan-related polymers: molecular structures of welan, S-657, and rhamsan

The primary structures of the four bacterial polysaccharides gellan, welan, S-657, and rhamsan are the same with respect to their backbones, but have different side-chains. This difference has a profound influence on their behavior in aqueous media. Solutions of gellan gum form stable aqueous gels under appropriate ionic conditions. By contrast, welan, S-657, and rhamsan do not gel but give very viscous solutions over a wide range of thermal, pH, and salt conditions. X-Ray fiber diffraction analysis and computer modeling of these branched polysaccharides demonstrate that they all have the same half-staggered, double-helical conformations as in the unbranched gellan, suggesting, therefore, that the side chains are responsible for diminishing gelling behavior. Depending on the size and location, the side chains shield the carboxylate groups to varying degrees; this shielding is substantial in welan and S-657, but less in rhamsan. In all cases, side-chain-main-chain interactions within the double helix prevent the carboxylate-mediated aggregation of double helices that is necessary for the gelation.     

Analysis of structure and function of gellans with different substitution patterns

Chemical mutagenesis or exposure to antibiotic stress of Sphingomonas paucimobilis ATCC 31461 and R40 have been used to isolate mutants producing modified gellan gum polysaccharides. N.m.r. and conventional carbohydrate analysis methods have been used to characterise these polysaccharides. The ¹H and ¹³C n.m.r. spectra of gellan gum have been fully assigned and the anomeric regions have been shown to be very sensitive to the type and location of non-carbohydrate substituents. Analysis of the gellan gum mutants suggests that they differ in the nature of acetate and glycerate substitution. Such gellan-related polysaccharides have been used to test the selective effect of acyl substituents on the gelation of gellan gum.     

Gelation behaviors of schizophyllan-sorbitol aqueous solutions

On addition of D-sorbitol, schizophyllan (SPG) aqueous solution forms a thermoreversible gel upon cooling. The gelation process is characterized by rheology, differential scanning calorimetry (DSC), and optical rotation measurement (ORD). It is found that the Winter-Chambon criterion works well in determining the critical gelation point of the present system, although the criterion has been scarcely applicable to systems that show weak-gel properties even before gelation. Moreover, ORD and DSC results indicate that a disordered to ordered conformational change accompanies the gelation process, which is attributed to the transition from SPG triple helix II to I. The gelation temperature of SPG-sorbitol aqueous solution is almost independent of SPG concentration in the examined concentration range and is slightly decreased by lowering SPG molecular weight, while greatly influenced by sorbitol content. The gelation is considered to be induced by the transition from SPG triple helix II to I, which leads to a three-dimensional network constituted by the extremely entangled and stiff SPG triple helices I. Furthermore, it is proved that neither junction zone nor aggregation of SPG triple helices is involved in the SPG-sorbitol gels. The SPG-sorbitol gel is structurally like a solution that is unable to flow within a timescale of usual observation.     

Structural modeling of snow flea antifreeze protein

The glycine-rich antifreeze protein recently discovered in snow fleas exhibits strong freezing point depression activity without significantly changing the melting point of its solution (thermal hysteresis). BLAST searches did not detect any protein with significant similarity in current databases. Based on its circular dichroism spectrum, discontinuities in its tripeptide repeat pattern, and intramolecular disulfide bonding, a detailed theoretical model is proposed for the 6.5-kDa isoform. In the model, the 81-residue protein is organized into a bundle of six short polyproline type II helices connected (with one exception) by proline-containing turns. This structure forms two sheets of three parallel helices, oriented antiparallel to each other. The central helices are particularly rich in glycines that facilitate backbone carbonyl-amide hydrogen bonding to four neighboring helices. The modeled structure has similarities to polyglycine II proposed by Crick and Rich in 1955 and is a close match to the polyproline type II antiparallel sheet structure determined by Traub in 1969 for (Pro-Gly-Gly)_n. Whereas the latter two structures are formed by intermolecular interactions, the snow flea antifreeze is stabilized by intramolecular interactions between the helices facilitated by the regularly spaced turns and disulfide bonds. Like several other antifreeze proteins, this modeled protein is amphipathic with a putative hydrophobic ice-binding face.     

Injectable in situ physically and chemically crosslinkable gellan hydrogel

An injectable, in situ physically and chemically crosslinkable gellan hydrogel is synthesized via gellan thiolation. The thiolation does not alter the gellan's unique 3-D conformation, but leads to a lower phase transition temperature under physiological conditions and stable chemical crosslinking. The synthesis and hydrogels are characterized by (1)H NMR, FT-IR, CD, or rheology measurements. The injectability and the tissue culture cell viability is also tested. The thiolated gellan hydrogel exhibits merits, such as ease for injection, quick gelation, lower gelling temperature, stable structure, and nontoxicity, which make it promising in biomedicine and bioengineering as an injectable hydrogel.     

Interpenetrating network formation in agarose–sodium gellan gel composites

Aqueous cold-set gels from mixtures of agarose and sodium gellan have been characterised structurally and mechanically using optical and electron microscopy, turbidity measurements, differential scanning calorimetry, mechanical spectroscopy and compression testing. Consistent with expectations for charged–uncharged polymer combinations at low ionic strength there is no liquid–liquid demixing in sols prior to gelation, and although transmission electron microscopy reveals heterogeneities in gel microstructures at the higher polymer concentrations, these are small in extent, and are unlikely to arise from normal segregative demixing. Overall, ‘molecularly’ interpenetrating networks (IPNs) are indicated, in which the gellan and agarose architectures pass through one another on a distance scale comparable to their pore sizes. At concentrations greater than 2% w/w gellan, where gellan is the first gelling species, and when the agarose concentration is greater than 0.5% w/w, the composite modulus falls below that expected for the agarose alone. At 0.5% w/w agarose, on the other hand, modulus contributions from the components are much closer to additive. These findings are reflected in the results of large deformation compression testing where breaking stresses show similar trends.     

Small globular protein motif forms particulate hydrogel under various pH conditions

Biocompatible hydrogels have great potentials in biomedical and biotechnological applications. In the current study, we reported a new naturally occurring protein motif that formed a transparent hydrogel when heated to 90 °C at a concentration as low as 0.4 mg/mL. The protein motif is the C-terminal soluble domain of an Escherichia coli inner membrane protein YajC (YajC-CT). We investigated the conformational change and self-assembly of the protein that lead to the formation of hydrogels using multiple methods. Atomic force microscopy studies of dilute gel samples revealed the presence of β-sheet-rich fibrils that were 2 to 3 nm in height and micrometers in length, which appeared to originate from homogeneous particles. On the basis of these observations, we proposed a three-step pathway of YajC-CT gelation. Hydrogels formed at different pH contained slightly different fibril structures. To our knowledge, this is the smallest hydrogel-forming globular protein module that has been characterized in detail. It may be useful as a model system in the elucidation of the mechanisms of protein fibrillation and gelation processes.     

Sphinganase, a new endoglycanase that cleaves specific members of the gellan family of polysaccharides.

A sporeforming gram-positive aerobic bacterium was isolated from soil and shown to secrete an endoglycanase that cleaves the tetrasaccharide backbone structure of specific members within the gellan family of related bacterial exopolysaccharides. We refer to these polysaccharides as sphingans. The structures of the sphingans differ by the type and position of side groups that are attached to the backbone. The new enzyme named sphinganase degrades welan, gellan, deacylated gellan, and polysaccharides S-88, S-7, and S-198. However, the enzyme does not attack rhamsan or polysaccharide NW11. Methods for growing the bacteria, isolating the enzyme, and assaying sphinganase activity are presented, and uses for the enzyme are proposed.     

On the sol-gel transition in solutions of Kappa-carrageenan

The disorder-order transition, which takes place at the gelpoint of κ-carrageenan solutions was monitored by optical rotation and light scattering measurements. The coincidence of both sets of experimental data affords good evidence that the sol-gel transition is accompanied by a conformational change. Transition temperatures were observed to be linearly dependent on the logarithm of the salt concentration and this result is explained by the formation of double helices.Heats of gelation were measured by differential scanning calorimetry. It was found that the enthalpy increases with ionic strength, which was ascribed to the occurrence of a secondary process in which double helices are assembled into larger aggregates.     

Rheological Study of the Gelation Process of Agarose-Based Solutions

The gelation of agarose is investigated by rheological methods and electron microscopy, as well as the thickening properties of xanthan. The gelling and thickening agents have been investigated in pure water to compare the results with theoretical models. The gelation of agarose was shown to follow two steps upon cooling, which could be addressed to the formation of helices and their aggregation. In addition to the rheology, transmission electron micrographs of freeze-dried samples have been taken to underline the date by corresponding structures at different stages of the gelling process. The xanthan molecules, which have been approximated by rigid highly charged rodlike molecules, undergo a jamming transition at a critical concentration. This concentration shows a strong dependence on the length of the molecules, which supports the high thickening effect of xanthan. When both, agarose and xanthan are mixed, the gel structure becomes very different. The gelling process is now determined by one step only. It is proposed that the jamming xanthan molecules prevent the formation of the aggregates of the agarose gel. The gels themselves appear then less elastic, and should yield a better mouth feeling.     

Identification and location of alpha-helices in mammalian cytochromes P450

A model of the alpha-helical structure of mammalian cytochromes P450 is proposed. The location and sequence of alpha-helices in mammalian cytochromes P450 were predicted from their homology with those of cytochrome P450cam, and these sequences were generally confirmed as helical in nature by using a secondary structure prediction method. These analyses were applied to 26 sequences in 6 gene families of cytochrome P450. Mammalian cytochromes P450 consist of approximately 100 amino acid residues more than cytochrome P450cam. This difference was accounted for by three major areas of insertion: (1) at the N-terminus, (2) between helices C and D and between helices D and E, and (3) between helices J and K. Insertion 1 has been suggested by others as a membrane anchoring sequence, but the apparent insertions at 2 and 3 are novel observations; it is suggested that they may be involved in the binding of cytochrome P450 reductase. Only the mitochondrial cytochrome P450 family appeared to show a major variation from this pattern, as insertion 2 was absent, replaced by an insertion between helices G and H and between helices H and I. This may reflect the difference in electron donor proteins that bind to members of this cytochrome P450 family. Other than these differences the model of mammalian cytochromes P450 proposed maintains the general structure of cytochrome P450cam as determined by its alpha-helical composition.     

Gellan gum biosynthetic enzymes in producing and nonproducing variants of Pseudomonas elodea.

A pathway for the synthesis of the repeating tetrasaccharide units in gellan gum from Pseudomonas elodea is proposed. The enzymes presumed to be involved in the synthesis of the activated precursors UDP-glucose, TDP-rhamnose, and UDP-glucuronic acid were detected and assayed in crude cell extracts of the gellan-producing (Gel+) P. elodea ATCC 31461. The levels of UDP-glucose pyrophosphorylase and TDP-glucose pyrophosphorylase were higher in cells grown in media leading to higher gellan yields. Moreover, these enzymes exhibited lower values in cells of a Gel- variant, spontaneously obtained from the Gel+ wild type. The activation or repression of their synthesis is thought to be involved in the expression of the mucoid phenotype. Nevertheless, based on results here reported, the involvement of other enzymes, that catalyze steps downstream from the formation of the precursors cannot be excluded.     

Gelation of gelatin observation in the bulk and at the air-water interface

Gelation of gelatin under various conditions has been followed by atomic force microscopy (AFM) with the objective of understanding more fully the structure formed during the gelation process. AFM images were obtained of the structures formed from both the bulk sol and in surface films during the onset of gelation. While gelation occurred in the bulk sol, the extent of helix formation was monitored by measurements of optical rotation, and the molecular aggregation was imaged by AFM. Interfacial gelatin films formed at the air-water interface were also studied. Measurements of surface tension and surface rheology were made periodically and Langmuir-Blodgett films were drawn from the interface to allow AFM imaging of the structure of the interfacial layer as a function of time. Structural studies reveal that at low levels of helical content the gelatin molecules assemble into aggregates containing short segments of dimensions comparable to those expected for gelatin triple helices. With time larger fibrous structures appear whose dimensions suggest that they are bundles of triple helices. As gelation proceeds, the number density of fibers increases at the expense of the smaller aggregates, eventually assembling into a fibrous network. The gel structure appears to be sensitive to the thermal history, and this is particularly important in determining the structure and properties of the interfacial films. © 1998 John Wiley & Sons, Inc. Biopoly 46: 245–252, 1998     

Crystal structure of a poly(rA) staggered zipper at acidic pH: evidence that adenine N1 protonation mediates parallel double helix formation

We have solved at 1.07 Å resolution the X-ray crystal structure of a polyriboadenylic acid (poly(rA)) parallel and continuous double helix. Fifty-nine years ago, double helices of poly(rA) were first proposed to form at acidic pH. Here, we show that 7-mer oligo(rA), i.e. rA₇, hybridizes and overlaps in all registers at pH 3.5 to form stacked double helices that span the crystal. Under these conditions, rA₇ forms well-ordered crystals, whereas rA₆ forms fragile crystalline-like structures, and rA₅, rA₈ and rA₁₁ fail to crystallize. Our findings support studies from ∼50 years ago: one showed using spectroscopic methods that duplex formation at pH 4.5 largely starts with rA₇ and begins to plateau with rA₈; another proposed a so-called ‘staggered zipper’ model in which oligo(rA) strands overlap in multiple registers to extend the helical duplex. While never shown, protonation of adenines at position N1 has been hypothesized to be critical for helix formation. Bond angles in our structure suggest that N1 is protonated on the adenines of every other rAMP−rAMP helix base pair. Our data offer new insights into poly(rA) duplex formation that may be useful in developing a pH sensor.     

Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili

We have used Escherichia coli as a model system to investigate the initiation of biofilm formation. Here, we demonstrate that E. coli forms biofilms on multiple abiotic surfaces in a nutrient-dependent fashion. In addition, we have isolated insertion mutations that render this organism defective in biofilm formation. One-half of these mutations was found to perturb normal flagellar function. Using defined fli , flh , mot and che alleles, we show that motility, but not chemotaxis, is critical for normal biofilm formation. Microscopic analyses of these mutants suggest that motility is important for both initial interaction with the surface and for movement along the surface. In addition, we present evidence that type I pili (harbouring the mannose-specific adhesin, FimH) are required for initial surface attachment and that mannose inhibits normal attachment. In light of the observations presented here, a working model is discussed that describes the roles of both motility and type I pili in biofilm development.     

Gelation of gellan gum aqueous solutions studied by polarization modulation spectroscopy

Circular birefringence (CB, or optical rotation) and linear birefringence (LB) were measured for gellan gum aqueous solutions with and without salt to examine the gelling system in the helical structure as well as in the orientation. It was found that gelling samples with salt show nonzero LB values, whereas LB is zero for the samples without salt even in the gel state. This difference can be explained by the thermal deformation of the system containing anisotropic aggregations of helices formed with the shielding effect of the added salt on the intramolecular and intermolecular electrostatic repulsions. Considering that the presence of LB in the system affects the estimation of CB, we developed an original procedure of the CB measurement to eliminate the contribution of LB. It was shown that our methods for eliminating the contribution of LB can improve the CB measurement for the gellan gum gel. The temperature dependence of [alpha] for the samples with salt in the gel state is quite different from that for the samples without salt, suggesting that the aggregates of helices in the samples containing a high concentration of salt form a supramolecular structure that contributes to CB.     

Interaction of water-soluble porphyrins with single- and double-stranded polyribonucleotides

CD and uv-visible absorption studies with several tetracationic water-soluble porphyrin derivatives show that some of these species can serve as probes to discriminate between A- and B-conformational forms of single-stranded polynucleotides. It is also observed that these porphyrins can participate in the formation of double helices by forming transient intermediate complexes enroute to duplex formation. © 1994 John Wiley & Sons, Inc.