A new bacterial polysaccharide (YAS34). I. Characterization of the conformations and conformational transition

This paper concerns the study of the conformational transition of a new exopolysaccharide (YAS34) using experimental techniques such as optical rotation, conductimetric and microcalorimetric measurements as a function of temperature. The behaviors of this polysaccharide in the acid or sodium salt form are compared; a deacetylated sample is also prepared to demonstrate the role of substituents. For the native structure (never heated), a conformational transition is observed but the deacetylated polysaccharide exhibits no ordered conformation. Multidetection size exclusion chromatography (SEC) analyses and conductimetric experiments allowed to determine the nature of each conformation and the molecular dimensions. From these results, it is suggested that the native conformation is a double helix which by heating over T(m) (temperature corresponding to half conformational transition) dissociates into disordered single chains. In the acid and sodium salt forms, by cooling below T(m), an ordered conformation is restored. This conformation seems to be an intramolecular double helix 'hairpin-like turn' (called renatured conformation). Nevertheless an irreversible denaturation is obtained progressively in the sodium salt form when the time of heating over T(m) increases. The conformation of the deacetylated polysaccharide corresponds to that of a single flexible chain (disordered conformation). The conformational transition for the native conformation was studied also in relation to the polyelectrolytic character of the polysaccharide: stability as a function of salt nature and salt and polymer concentrations was investigated for the polymer initially in the sodium and acid forms.     

Structure and properties of a bacterial polysaccharide from a Klebsiella strain (ATCC 12657)

The chemical structure and the rheological behavior of the Klebsiella polysaccharide ATCC 12657 was studied and compared with data described in the literature and obtained for similar polysaccharides. The acetylated polysaccharide presents in solution a normal viscoelastic behavior with no evidence of an ordered conformation whatever the experimental conditions are. The deacetylated form can induce the formation of physical gels, in the presence of salt excess or ethanol. Microcalorimetry, optical rotation, and rheology experiments demonstrate that a thermally reversible and highly cooperative conformational transition occurs at the same temperature than a sol-gel transition. The melting of the gel and the conformational transition temperatures are dependent on the nature of cations and ionic concentration, whereas the gel strength is only influenced by polymer concentration.     

Revisiting the conformation of xanthan and the effect of industrially relevant treatments

The structure and conformation of xanthan in aqueous solution following various processing treatments typically encountered in its application were investigated in this study. Treatments such as heating, autoclaving, high pressure homogenisation and irradiation were subjected to the same sample. Parameters such as weight average molecular weight (M(w)), polydispersity index, root mean square radius of gyration, intrinsic viscosity and Huggins constant were used to monitor the effect of these treatments. Additionally, we have quantified the mass recovery of samples examined by gel permeation chromatography and light scattering to properly account for all fractions present in xanthan solutions. Atomic force microscopy (AFM) images together with height measurements confirmed that xanthan conformation is double helical ordered renatured state (pre-heat treated by the manufacturer) in dilute solution conditions and random coil conformation in very dilute solution. The ordered (renatured) conformation is shown to have partially molten double helix, with more flexibility than the perfectly ordered native double helix. Heat treatment for 2h at 85°C reduces the M(w) of xanthan to half its initial value, and mass recovery measurements indicate that it completely overcomes its associative nature. Thermally treated xanthan solution in the dilute region leads to an order-disorder transition, as determined by contour length per unit mass. Similarly, irradiation of xanthan solution results in an order-disorder transition together with the production of single strand low molecular weight molecules. Autoclaving and high pressure homogenisation treatments cause degradation of xanthan. The results from treated xanthan solutions following high pressure homogenisation and irradiation confirm that xanthan does not reassociate. A revised summary of xanthan conformation in solution together with schematic models following the various treatments are proposed.     

Competitive inhibition of interchain interactions in polysaccharide systems

The scope and validity of competitive inhibition to explore specific intermolecular association in polysaccharide systems is demonstrated, using alginate, carrageenan and xanthan-galactomannan gels as models. Alginate gelation can be abolished almost completely in the presence of an equimolar concentration of short poly-l-guluronate chain segments, and weakened by short mixed sequences, while poly-d-mannuronate has little effect; this is consistent with previous evidence of the relative importance of these three block-types in interchain association. Carrageenan gels are weakened by the presence of short homologous segments only under conditions of limited helix-helix aggregation, where it is understood that direct interchain association through double helices contributes appreciably to total crosslinking. Gel formation by association of the ordered conformation of xanthan with poorly substituted mannan backbone regions of galactomannans is significantly inhibited by the addition of a galactomannan having fewer unsubstituted chain sequences, and which can therefore bind to xanthan without significant network formation. In general, therefore, gel formation through ordered junction zones of fixed stoichiometry is inhibited by chains that are capable of participation in one stable junction, but not in two or more, so that they occupy binding sites without contributing to the development of the network.     

Influence of the charge density on the solution behaviour of polycarboxylates derived from the polysaccharide scleroglucan

Solution properties of polycarboxylates obtained by periodate oxidation of scleroglucan, with different degrees of oxidation, have been studied as a function of pH. Viscosity, calorimetric, optical and chiro-optical measurements show that the two carboxylated samples with higher degrees of oxidation (S-1·0 and S-0·7) can assume an ordered conformation at low pH. On the contrary the sample characterized by a lower extent of oxidation (S-0·2) shows no conformational changes. Optical measurements suggest that this polymer would be in an ordered helical form in dilute aqueous solution.     

Role of substituents on the properties of some polysaccharides

This paper concerns the influence of the chemical structure on the physical properties of some polysaccharides. Especially, we proposed to discuss the role of the substituents on these properties. In some cases, non-carbohydrate substituents play a minor role on rheological properties in the presence of a salt excess as shown on xanthan and succinoglycan. The rheology of aqueous solution of these stereoregular polysaccharides is controlled by the conformation (helical conformation) whose stability is not largely influenced by these substituents. On the other hand, the interaction between galactomannan and xanthan depends on the presence of acetyl substituents on xanthan but also on the xanthan conformation. However, for polymers such as gellan, XM-6 or BEC 1615, complete deacetylation induces the ability to form physical gels in given thermodynamic conditions. The presence of carbohydrate substituents or short side chains was also examined. Especially in the gellan family, the role of position of substitution (position 3 on the glucose unit C or position 6 on the A glucose) was presented. It is concluded that the substituents giving the higher stability for the helical conformation (higher DeltaH and Tm values) also cause a lower salt sensitivity for the helical stability. The role of the substituents on the properties is also described for natural polymers and their chemically or enzymatically modified derivatives.     

Electron microscopy of native xanthan and xanthan exposed to low ionic strength

Optical rotation data indicate that xanthan can exist both in an ordered and a disordered conformation. Using molecular weights obtained from light scattering measurements and contour length distributions obtained from electron micrographs, we find that a native, filtered xanthan exposed to low salinity (< 10^?4M NaCl) and subsequently returned to 0.1M NaCl has a highly elongated structure with a mass per unit length of 1950 ± 200 Dalton/nm. Our data thus suggest that the ordered conformation of this xanthan is double stranded. We find that native, filtered xanthan in 0.1M NH₄Ac has a nearly similar structure, but exists in part as aggregates of varying shape and size. Electron micrographs of these xanthans in 10^?4M NH₄Ac (the disordered conformation) display a mixture of species ranging from unaggregated single- or perfectly matched double-stranded species, to double-stranded chains branching into its two subunits as well as double-stranded chains with different degrees of mismatching. This study suggests that the perfectly matched antiparallel or parallel double-stranded chain constitutes the lowest free energy state of the ordered conformation of xanthan in dilute aqueous solution.     

Relationships between conformation of beta-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of beta-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of beta-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon beta-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding beta-sheet structure as evidenced by the appearance of a strong band at 1614 cm(-1) at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of beta-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular beta-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of beta-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular beta-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.     

Reexamining the egg-box model in calcium-alginate gels with X-ray diffraction

The structure of the Ca--alginate junction zones was investigated with X-ray scattering on gels prepared with different methods. Fiber diffraction reveals the popular egg-box model may not be the only possible structure for the junction zones. The (001) reflection, which should be extinguished due to 2/1 helical conformation in the egg-box model, was observed. This was further confirmed by the measurements on Ca--alginate gel beads prepared at different pH where large pieces were formed through a relatively slow process, which leads to a higher crystallinity and a more perfect ordering. The results suggest a 3/1 helical conformation is more proper for Ca--alginate gels formed slowly. This does not exclude the possibility for the 2/1 helical conformation in fast gelatinized Ca--alginate in which the 2/1 helix is a metastable form. Comparing the X-ray scattering results of the fresh, dehydrated, and rehydrated gels, a reversible aggregation of junction zones is found during dehydration and rehydration. The different stabilities of initial bonds and bonds formed during drying are speculated to be the contribution of MG block or short G blocks in the junction zones.     

CD conformational studies on synthetic peptides encompassing the processing domain of the ocytocin/neurophysin precursor

Synthetic peptides of different size, reproducing the proteolytic processing site of proocytocin, were studied by CD under several experimental conditions in order to ascertain the ability of different solvents to stabilize secondary structural motifs, such as α-helix tracts and β-turns. A combination of deconvolution methods and empirical calculations subtracting the contributions due to unordered structures from the spectra suggests that in solution (a) mainly two distinct families of ordered conformers containing structurally different β-turns are present, (b) the relative stability of the different conformers depends from the nature of the solvent, and (c) in the case of the larger peptides, a population containing an α-helical conformation is also present. From the biological point of view the presence of at least two families of ordered conformers could be in line with current theories assuming that the catalytic effect of the receptor microenvironment may be determinant in shifting the equilibrium toward the active conformation. © 1997 John Wiley & Sons, Inc. Biopoly 41 : 461–479, 1997     

On the physicochemical properties of gellan gum

This paper concerns the behavior in dilute and demidilute solutions of deacetylated gellan. The conformational transition, controlled by temperature and ionic strength, is investigated. It corresponds to a double-helix single-chain transition. Large ionic selectivity is observed in the helical conformation th at controls the degree of aggregation upon gelation. Potentiometry and conductivity measurements are interpreted in terms of the Manning polyelectrolyte theory in the sol state.     

Salt-induced extension and dissociation of a native double-stranded xanthan

This study shows that xanthan molecules at room temperature may assume at least three different conformations in 0.1 m NaCl aqueous solutions in which the local structure is ordered: (1) the native compact double helix, (2) the extended double helix, and (3) the extended single helix. Experiments including viscosity, low-angle light scattering and optical rotation measurements have been carried out with a fully pyruvated and fully acetylated native laboratory sample supplied as fermentation broth. Two major conformation changes of the native double helix which were found irreversible in our experimental conditions can be induced by treatments at low ionic strength. After treatment in 10⁻⁴m NaCl, xanthan is still a double helix in 10⁻¹m NaCl, but the backbone of each strand has been extended. After the sample has been in 10⁻⁵m NaCl, the double helix has been dissociated and a single helix sample is obtained. Thus, the denaturing of xanthan is a two-step process. The first step consists of the extension of the two chains inside the double helix, and the second is a dissociation of the native double strand.     

A potentiometric and CD investigation on the conformational properties of poly (L-p-aminophenylalanine) in aqueous solution

The conformational properties of poly(L -p-aminophenylalanine) have been investigated by potentiometric and circular dichroism (CD) techniques. It has been found that the polymer in the charge-free state can assume two ordered conformations, depending upon temperature conditions. At room temperature the polymer assumes the right-handed helical form by described Goodman and Peggion.¹ At temperatures higher than 40°C a new ordered conformation has been found, whose slow rate of formation and ir absorption properties are typical of the β-structure. The thermodynamic parameters relative to the coil-β transition, determined by potentiometric titration techniques, revealed that the thermodynamic stability of the β-structure is mainly due to enthalpic factors. The formation of this structure is unfavored on a kinetic basis.     

Insights into prion biology: Integrating a protein misfolding pathway with its cellular environment

Protein misfolding and assembly into ordered, self-templating aggregates (amyloid) has emerged as a novel mechanism for regulating protein function. For a subclass of amyloidogenic proteins known as prions, this process induces transmissible changes in normal cellular physiology, ranging from neurodegenerative disease in animals and humans to new traits in fungi. The severity and stability of these altered phenotypic states can be attenuated by the conformation or amino-acid sequence of the prion, but in most of these cases, the protein retains the ability to form amyloid in vitro. Thus, our ability to link amyloid formation in vitro with its biological consequences in vivo remains a challenge. In two recent studies, we have begun to address this disconnect by assessing the effects of the cellular environment on traits associated with the misfolding of the yeast prion Sup35. Remarkably, the effects of quality control pathways and of limitations on protein transfer in vivo amplify the effects of even slight differences in the efficiency of Sup35 misfolding, leading to dramatic changes in the associated phenotype. Together, our studies suggest that the interplay between protein misfolding pathways and their cellular context is a crucial contributor to prion biology.Key words: prion, protein misfolding, chaperones, amyloid, ordered aggregates, transmission, aggregate size, Sup35, Hsp104     

Size Distribution and Molecular Associations of Plasma Fibronectin and Fibronectin Crosslinked by Transglutaminase 2

Fibronectin (FN) is a ubiquitously expressed cell adhesion protein capable of assembling into large, extended fibrillar networks as part of an extracellular matrix (ECM) that regulates cell behavior. FN is a substrate for certain members of the transglutaminase family of protein-crosslinking enzymes-enzymes which can modify the ability of FN to support cell adhesion. In this study, we have analyzed the thermo-chemical stability of plasma FN in its noncrosslinked form, and after crosslinking by transglutaminase 2 (TG2), using dynamic light scattering. We report that FN is found in a generally globular (8.7 nm hydrodynamic radius), dimerized form in aqueous solutions, but unfolds into a linear arrangement at high ionic (1 M NaCl) and chaotropic (5 M urea) environments. FN conformation remained stable after multiple heating and cooling cycles ranging from 4 to 60 degrees C. Crosslinking of FN with TG2 formed large, multimeric complexes having high chemical stability in aqueous, high ionic and chaotropic environments, demonstrating that this covalent modification stabilizes FN. Given recent data that substrate (e.g. ECM) rigidity profoundly affects cell differentiation and behavior, we further studied how TG2 crosslinking affects the molecular rigidity of FN by obtaining atomic force microscopy nanoindentation measurements from untreated and crosslinked FN samples embedded in acrylamide gels. We demonstrate that TG2-mediated crosslinking of FN significantly increases Young's modulus (of elasticity), an observation of increased rigidity having important implications with respect to the biological role of ECM protein-crosslinking in cell signaling and guiding cell differentiation.     

Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of β-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of β-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon β-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding β-sheet structure as evidenced by the appearance of a strong band at 1614 cm⁻¹ at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of β-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular β-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of β-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular β-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.     

GDP state of tubulin: stabilization of double rings

Purified tubulin, with GDP occupying the exchangeable nucleotide binding site, has been examined conformationally and for its ability to self-associate into double rings. The circular dichroism spectrum increased by ca. 10% in negative amplitude between 205 and 225 nm over the spectrum of tubulin in the GTP state, but there were no significant shape changes. This indicates that replacement of GTP by GDP induces tubulin to adopt a more ordered conformation. The sedimentation coefficients of tubulin alpha-beta dimers in the GDP and GTP states were identical, with s20,w = 5.8 S. A sedimentation velocity study of tubulin in the GDP state showed that, in the presence of magnesium ions, this protein undergoes a reversible Gilbert-type self-association. The end product of this reaction was found to be 26 subunit double rings identical with those described by Frigon and Timasheff [(1975) Biochemistry 14, 4567-4599] for a similar polymerization of tubulin in the GTP state. Analysis of the data showed that Tu-GDP has a much stronger propensity for the formation of double rings than Tu-GTP, the corresponding equilibrium with constants for the 26Tu in equilibrium Tu26 being 4.2 X 10(119) M-25 and 2.27 X 10(109) M-25 for Tu-GDP and Tu-GTP, respectively. This leads to Tu-GTP being predominantly in the form of alpha-beta dimers and Tu-GDP in the form of double rings under normal experimental conditions used in the study of microtubule assembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Author index to volume 175

500 MHz ¹H-NMR and NOE measurements of d(GGATCC) and d(GGm⁶ATCC) show that both oligo-nucleotides assume a B-DNA conformation at low temperature. Around the melting temperature, however, the single and double strands of the N-methylated form are in slow exchange on the NMR time scale. The preferred conformation of the adenine methyl group, cis to N¹, retards base pairing and also destabilizes the double helix.     

Studies on the conformation of protonated DNA

Experiments were made to demonstrate the predominant protonation effects and structural changes of the ordered double helical DNA structure and denatured state of DNA. Spectrophotometric titrations performed at different wavelengths indicate that cytosine can be protonated in the DNA double helical molecule to a high extent without breakdown of the secondary structure. With DNA heat-denatured under severe conditions the protonation of cytosine can be measured at 280, 295, and 300 mμ: the apparent pK value obtained was ～4.6. The protonated double helical conformation of the DNA molecule differs from the unprotonated state, which follows from the decrease of the thermal stability and from changes in the ORD curves. The ORD of a GC-rich DNA indicates a novel Cotton effect with positive rotations at ～260 mμ in 0.02M KCl below pH 4.0 to pH 3.3. The occurrence of the new peak parallels the extent of protonated cytosine measured by the spectrophotometric titrations. It is concluded that the protonated cytosine in the double helical structure is responsible for the difference between the protonated DNA conformation and the native state at neutral pH.     

Conformational investigation on the bacterial polysaccharide xanthan

The conformation of xanthan has been investigated as a function of temperature, ionic strength, and polymer concentration. A reversible transition induced by temperature is demonstrated; the melting temperature (T_M) is directly correlated to the total ionic-strength and is independent of the polymer concentration. Measurements of circular dichroism show that the polysaccharide exists in a combination of only two characteristic conformations (random and ordered), regardless of the temperature and the concentrations of salt and polymer. Hydrodynamic measurements show that the hydrodynamic volume of both conformations is almost constant over the range of temperature investigated. The mechanism proposed by Morris for melting is confirmed, and a multichain process is excluded. The birefringence stability of the concentrated solutions is discussed.