Induction of metachromasia and circular dichroism in the dye 1,9-dimethyl methylene blue by S. aureus wall teichoic acid

Teichoic acid (TA) isolated from the Gram-positive bacteria Staphylococcus aureus induces blue shifted metachromasia in the dye 1,9-dimethyl methyleneblue (DMMB). Results of conductometric titrations show that DMMB binds to TA with 1:1 stoichiometry. Unlike DNA, this TA does not induce a definite metachromatic band in the dye acridine orange. S. aureus TA also induces strong circular dichroism (CD) in this dye with molar ellipticity value of the order of 10(4). This induction of CD in DMMB supports our earlier inference that TA in dilute solution at neutral pH has a helical conformation, and is not a random coil polymer.     

Light scattering studies of solutions of the bacterial polysaccharide (XM6) elaborated by Enterobacter (NCIB 11870)

Light scattering studies have been made on solutions of the sodium and tetra-methyl ammonium salts of the anionic heteropolysaccharide XM6. The polymers may be modelled as stiff wormlike chains. In dilute solution no conformational change was observed upon increasing the ionic strength. Models for gelation at higher polymer concentration are discussed. The measured mass per unit length of the polymer favours an ordered helical conformation and a gelation mechanism involving association and possibly crystallization of segments of the helical chains.     

Structure of amphotericin B aggregates based on calculations of optical spectra

The Degenerate ground state approximation was used to calculate the optical absorption and CD spectra for helical polymer models of amphotericin B aggregates in aqueous solution. Comparisons with experimental spectra indicate that a two-molecule/unit cell helical polymer model is a possible structure for aggregates of amphotericin B.     

Far-ultraviolet optical activity of saccharide derivatives. II. Dextran,amylose, and mycodextran acetes; dextran and amylose xanthates

The ultraviolet ORD and CD spectra of amylose, dextran, and mycodextran acetates and some of thier oligomers were recorded in trifluoroethanol solution in the 300–185nm wavelength range. Similarly, the spectra of amylose and dextran xanthates in water solution were obtained in the 400–200 nm range. In the amylose acetate series, the monomer and dimer both show a normal acetyl n → π* transition in CD, while the trimer and the polymer both exhibit an additional, shorter wavelength peak. The latter is presumed to arise from a helical conformation of the amylose chain. This interpretation is substantiated by a helix–coil type transition of the CD spectra of amylose triacetate at elevated temperatures and a reversion of the anomalous CD to the normal CD upon partial deacetylation. By contrast, neither dextran acetates nor mycodextran acetate exhibit any conformational effects. The CD of dextran acetates is quite sensitive to β-1,6 and branch linkages. The ORD and CD of amylose xanthate are complex, suggesting the presence of organized structure in solution. The dextran xanthate shows only a simple ORD spectrum and no observable CD.     

Interaction of the ruthenium red cation with nucleic acid double helices

The hexapositive complex cation ruthenium red very effectively stabilizes DNA and RNA double helices against thermal denaturation. In the presence of nucleic acid helices, this symmetric cation acquires an extrinsic CD spectrum near the wavelength of the dye's maximum absorbance. Competition experiments with single-stranded polyd(T) show this induced CD to be the result of selective binding to helical sites. The preferential affinity of ruthenium red for double helical binding sites is so great that it brings about biphasic absorbance- temperature profiles of polyd(A-T) at low [cation]: [polynucleotide phosphate]. The visible CD signal and fraction of helix melting at the upper transition increases with ruthenium red concentration until approximate charge neutrality is reached. These interactions, which have been studied in detail with the poly(U-U) helix as well as polyd(A-T), are likely largely electrostatic, since sufficient [NaCl] eliminates the bipliasic melting of polyd(A-T), renders the ultraviolet absorbance of poly (U) insensitive to ruthenium red, and abolishes the induced CD effects. The bipliasic melting of polyd(A-T) at intermediate [dye] is attributed to saturation of remaining double helical segments by cation migration from newly melted regions- Furthermore, virtually no change was observed in the induced CD upon melting through the first transition, whereas the effect is destroyed upon inciting through the second transition. A quantitative treatment of the data is used to obtain binding site size and association constant for the complex. The induced effect may prove useful in the exploration of exposed nucleic acid helical structure in such complex particles as nucleosomes or ribosomes.     

Fabrication and structural characterization of the Langmuir–Blodgett films from a new chitosan derivative containing cinnamate chromophores

A new amphiphilic chitosan derivative, octanoylchitosan cinnamate (OCC) was synthesized through regioselective modifications of chitosan. A solution of OCC was spread to water to form a stable monolayer at the air/water interface. The surface pressure (π)–area (A) isotherm indicated that the polymer had a limiting area of about 100 Å² per repeat unit. YZ-type multilayers were deposited onto hydrophobic substrates through Langmuir–Blodgett (LB) technique. The structural features of the LB films were investigated by UV absorption, circular dichroism (CD) and linear dichroism (LD) spectroscopy. The results showed that the intrinsic chirality originating from the helical order of the OCC backbones was maintained in the LB films. Besides, the polymer backbones were uni-axially oriented in the LB film. The ordered structures of OCC assembled in a dilute solution and in a cast film were also investigated and the results were compared with that of the LB film.     

Recovery of Clostridium thermosulfurogenes produced beta-amylase by (hydroxypropyl)methylcellulose partition.

A procedure for recovering Clostridium thermosulfurogenes produced beta-amylase from fermentation broth by partition was developed. The partition was achieved by addition of ammonium sulfate to an aqueous solution of the enzyme with (hydroxypropyl)methylcellulose. The beta-amylase-containing pellet formed upon centrifugation could be redissolved and the polymer recovered by a second salt addition. The process was not dependent on polymer/enzyme solution pH, but it was affected by temperature, polymer nominal molecular weight and loading, and fermentation carbon source. Unlike more traditional aqueous-phase partitions, such as poly(ethylene glycol)/dextran, the current approach appeared to be biospecific.     

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.     

Helical screw sense bias in chiral polyfluorene stimulated by solvent

Conjugated homopolymer poly(9,9‐bis(3‐((S )‐2‐methylbutylpropanoate))fluorene) (PSF) with chiral pendants was synthesized and characterized. Dissolution experiments show that PSF is well dissolved in racemic limonene at high temperature and begins aggregating upon sequential cooling treatment. The corresponding assemblies were transferred to quartz plate by the spin‐coating method. Comparably, film casting from chloroform solution was also prepared. Upon annealing thermal treatments, these PSF films exhibited perfect mirror circular dichroism (CD) Cotton effects and dissymmetry ratios. Optical absorption spectroscopy (UV‐vis), CD, and fluorescence spectroscopy results reveal that chiral side chains successfully induced M ‐ and P ‐helical structures in aggregates and films, and this significant difference was ascribed to their differential supramolecular conformations.     

A new bacterial exopolysaccharide (YAS34). II. Influence of thermal treatments on the conformation and structure. Relation with gelation ability

This paper describes an original mechanism of evolution of a polysaccharide system occurring during thermal treatments. Under its native conformation, the YAS34 polymer presents a solution character in the dilute and semi-dilute regimes. However, the zero shear rate viscosity indicates existence of interchain interactions which disappear on the deacetylated polymer. Thermal treatments over the temperature of conformational change produce a progressive and irreversible evolution of the physical properties when the polymer is under its sodium salt form. This evolution was related to a modification of the arrangement of acetyl substituents. The heated polysaccharide gives thermoreversible gel which is very elastic. A gelation mechanism is proposed based on formation of helical segments connecting the network.     

Toroidal condensation of Z DNA and identification of an intermediate in the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC)

Using a combination of spectroscopic techniques, quasi-elastic laser light scattering (QLS), and electron microscopy (EM), we have been able to show that the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC) is accompanied by extensive condensation of the DNA in both low and high ionic strength buffers. At low concentrations of NaCl (2 mM Na+), an intermediate rodlike form, which exhibits a circular dichroism (CD) spectrum characteristic of an equimolar mixture of B and Z forms, is observed. This is produced by the orderly self-association of about four molecules of the polymer after prolonged incubation of a concentrated solution at 4 degrees C. On addition of 5 microM Co(NH3)63+, the CD spectrum of the intermediate changes to that of the Z form, which is visualized as a dense population of discrete toroids on an EM grid stained with uranyl acetate. On the other hand, addition of NaCl to a solution of poly(dG-m5dC) X poly(dG-m5dC) in the absence of any multivalent ion condenses the polymer to toroidal structures at the midpoint (0.75 M NaCl) of the B to Z transition. Further addition of NaCl unfolds these toroids to rodlike structures, which show characteristic Z-form CD spectra. These results show that Z DNA can take up a variety of tertiary structural forms and indicate that its inverted CD spectrum is due to its left-handed helical sense rather than to differential scattering artifacts.     

The effect of simple shear flow on the helix–coil transition of poly‐L‐lysine

Studies of the helix‐to‐coil transition in dilute solutions of poly‐L ‐lysine, dissolved in mixtures of water and methanol (MeOH), have been carried under shear flow using flow birefringence and modulated polarimetry. The fraction of helical conformations in a given solution remains independent of shear rate for MeOH concentrations above and below the critical value for the helix‐coil transition (i.e., 87.5% MeOH). For the 87.5% MeOH solutions, a shear‐induced helix‐to‐“stretched” coil transition occurs above a critical shear rate. Induction times for the transition show a temperature and shear rate dependence that can be described in terms of an activated jump process. Measurements of circular birefringence on cessation of flow also show that the transition is reversible, with the stretched coil reverting to the helical state on a time scale of several seconds. The activation energy for the jump process is found to be 16.2 kJ/mole. © 1999 John Wiley & Sons, Inc. Biopoly 50: 589–594, 1999     

Physical properties of poly (3,N4-ethenocytidylic acid)

Homopolymers of etheno CMP have been prepared by the action of polyribonucleotide phosphorylase upon etheno CDP. At alkaline pH the optical properties are consistent with a structure consisting of partially helical single-stranded chains whose helical regions are stabilized by base stacking. At acid pH the degree of helicity increases markedly. The degree of cooperativity displayed by the helix leads to coil transition induced by pH or temperature is less than for the case of polyribocytidylic acid. In the presence of acridine orange the alkaline form develops a strong extrinsic CD spectrum.     

Preparation and chiro-optical characterization of polyaniline doped with (+) or (-)-2-pyrrolidone-5-carboxylic acid (PCA)

Optically active polyaniline (PANI) salts were readily generated in solution via the enantioselective acid doping of neutral emeraldine base (EB) form of PANI with either (+) or (-)-2-pyrrolidone-5-carboxylic acid (PCA) in dimethylsulfoxide (DMSO) and dimethylformamide (DMF) solvents. Strong mirror imaged circular dichroism (CD) spectra were obtained for the deep green polymer solutions obtained with (+) or (-) PCA, suggesting that the acid doping is enantioselective, with one helical screw of the polymer chain being preferentially produced depending on the nature of enantiomer. It was observed that molar concentration of PCA as well as nature of solvent plays a very important role in the generation of optically active PANI. The generated optically active PANI did not show any loss of optical activity up to 200 h.     

Affinity thermoprecipitation of trypsin using soybean trypsin inhibitor conjugated with a thermo-reactive polymer,poly(N-vinyl caprolactam)

The soybean trypsin inhibitor conjugate with a thermo-reactive water soluble polymer, poly(N-vinyl caprolactam), was successfully used for thermally induced affinity precipitation of trypsin. The validity of the developed procedure was proven by a model separation of trypsin from dilute solution containing a large excess of bovine serum albumin.     

Optical investigations on double stranded ribonucleic acid from turnip yellow mosaic virus

Summary TYMV-dsRNA has been studied by ORD and CD. The spectra indicate a highly ordered secondary structure which disappears upon heating or titrating to acid pH. No distinct acid structure as found for DNA could be observed.Melting profiles of TYMV-dsRNA show a strong dependence on ionic strength which is analogous to synthetic polynucleotide complexes. Upon storage for several months the melting points drop considerably, without change in the ORD spectra. The two sets of melting points obtained for TYMV-dsRNA compare well with the results obtained in the literature: two series of melting points have been found for ds-RNA of a wide variety of origins. If plotted as a function of their G-C content, these data fall on two different lines with different slopes.It is suggested that ds-RNA might exist in two different helical forms and that TYMV-dsRNA might undergo a conformational change in solution upon storage.Boursier du Commissariat à l'Energie Atomique.     

Insight into the mechanism of internalization of the cell-penetrating carrier peptide Pep-1 through conformational analysis

Recently, we described a new strategy for the delivery of proteins and peptides into mammalian cells, based on an amphipathic peptide of 21 residues, Pep-1, which was designed on the basis of a protein-interacting domain associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of active proteins or peptides both in cultured cells and in vivo, without requiring any covalent coupling. We have examined the conformational states of Pep-1 in its free form and complexed with a cargo peptide and have investigated their ability to interact with phospholipids and the structural consequences of these interactions. From the conformational point of view, Pep-1 behaves significantly differently from other similarly designed cell-penetrating peptides. CD analysis revealed a transition from a nonstructured to a helical conformation upon increase of the concentration. Determination of the structure by NMR showed that in water, its alpha-helical domain extends from residues 4-13. CD and FTIR indicate that Pep-1 adopts a helical conformation in the presence of phospholipids. Adsorption measurements performed at the air-water interface are consistent with the helical form. Pep-1 does not undergo conformational changes upon formation of a particle with a cargo peptide. In contrast, we observe a partial conformational transition when the complex encounters phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Conformational change of Pep-1 is not associated with complexation with its cargo but is induced upon association with the cell membrane.     

Biosynthesis of an amphiphilic silk-like polymer

An amphiphilic silk-like protein polymer was efficiently produced in the yeast Pichia pastoris. The secreted product was fully intact and was purified by solubilization in formic acid and subsequent precipitation of denatured host proteins upon dilution with water. In aqueous alkaline solution, the negatively charged acidic polymer assumed extended helical (silk III-like) and unordered conformations. Upon subsequent drying, it assumed a conformation rich in beta-turns. In water at low pH, the uncharged polymer aggregated and the solution became turbid. Concentrated solutions in 70% (v/v) formic acid slowly formed gels. Replacement of the formic acid-water mixture with methanol and subsequent drying resulted in beta-sheets, which stacked into fibril-like structures. The novel polymer instantaneously lowered the air-water interfacial tension under neutral to alkaline conditions and reversed the polarity of hydrophobic and hydrophilic solid surfaces upon adsorption.     

Measurement of the secondary structure of adsorbed protein by circular dichroism. 1. Measurements of the helix content of adsorbed melittin.

A new circular dichroism (CD) technique is presented which quantifies, in situ, the changes in protein and peptide secondary structure upon adsorption at the quartz/liquid interface. Far-UV CD spectra of adsorbed proteins were recorded from several quartz interfaces contained in a specially constructed cell. Adsorbed, oriented alpha-helical spectra were recorded from hydrophilic and hydrophobic quartz using the bee venom peptide, melittin, which can be induced into an alpha-helical, tetrameric conformation in solution. The hydrophobic quartz provides a model system for oil-in-water emulsions and cell membranes. Surface concentrations were determined by radio-counting and were dependent on the nature of the surface. The characterization of these spectra has been partly achieved using far-UV CD spectra obtained from melittin adsorbed onto hydrophilic colloidal silica particles, where orientation effects are eliminated. Analysis of these spectra reveals considerable denaturation of the helical structures upon adsorption. Surface concentrations from the silica were determined from adsorption isotherms. The surface orientation of adsorbed melittin was dependent on the state of aggregation and hence degree of helicity of the molecule. These results support a model for the mode of action of melittin in lysing membranes.     

Conformational transition of DNA induced by cationic lipid vesicle in acidic solution: spectroscopy investigation

The conformational transition of DNA induced by the interaction between DNA and a cationic lipid vesicle, didodecyldimethylammonium bromide (DDAB), had been investigated by circular dichroism (CD) and UV spectroscopy methods. We used singular value decomposition least squares method (SVDLS) to analyze the experimental CD spectra. Although pH value influenced the conformation of DNA in solution, the results showed that upon binding to double helical DNA, positively charged liposomes induced a conformational transition of DNA molecules from the native B-form to more compact conformations. At the same time, no obvious conformational changes occurred at single-strand DNA (ssDNA). While the cationic lipid vesicles and double-strand DNA (dsDNA) were mixed at a high molar ratio of DDAB vesicles to dsDNA, the conformation of dsDNA transformed from the B-form to the C-form resulting in an increase in duplex stability (DeltaT(m)=8+/-0.4 degrees C). An increasing in T(m) was also observed while the cationic lipid vesicles interacted with ssDNA.