Melting Of Helices Research Articles

Effect of the number of nucleic acid oligomer charges on the salt dependence of stability (DeltaG 37degrees) and melting temperature (Tm): NLPB analysis of experimental data.

For nucleic acid oligomers with variable chain lengths, the salt concentration ([salt]) dependences of the denaturation temperature (T(m)) and of the free energy of helix formation at 37 degrees C (Delta) are predicted using nonlinear Poisson-Boltzmann (NLPB) calculations. Analysis of experimental data reveals that the ratio of the [salt] derivative of melting temperature (ST(m) = dT(m)/d log[salt]) to the value for a polymer with the same base composition (ST(m)/ST(m, infinity)) is independent of base composition but strongly dependent on the number of DNA charges (/Z/) below approximately 8 bp for two-strand helices (formed from association of two complementary strands) and below approximately 18 bp for hairpin helices (formed from folding of one self-complementary strand). We interpret these ST(m)/ST(m, infinity) ratios in terms of the ratio of thermodynamic ion release from the oligomer (Deltan(u), per charge) to that from the same oligomer embedded in polymeric DNA (Deltan(u, infinity), per charge). Experimental values of ST(m)/ST(m, infinity) and its dependence on /Z/ are in good agreement with NLPB predictions for a preaveraged (essential structural) model of DNA. In particular, the NLPB calculations describe the stronger /Z/ dependence of ST(m) observed for melting of oligomeric hairpin helices than for melting of two-strand helices. These calculations predict an experimentally detectable (>or=10%) difference between ST(m) and ST(m, infinity) which increases strongly with decreasing length for two-strand helix lengths of <15 bp and for hairpin helix lengths of <30 bp. From NLPB values of Deltan(u)/Deltan(u, infinity), we predict Delta as a function of [salt] and /Z/. Predictions of thermodynamic and thermal stabilities of oligomeric helices as functions of length and [salt] are consistent with and represent a significant refinement of the average oligomer salt effect currently in use in nearest neighbor stability predictions.

Role of cosolutes in gelation of high-methoxy pectin. Part 2. Anomalous behaviour of fructose: calorimetric evidence of site-binding

Gels of high-methoxy pectin (DE 70; 1.0 wt%; pH 3.0) in the presence of fructose at concentrations of 50, 55, 60 and 65 wt% showed an intense endotherm followed immediately by an intense exotherm on heating. These transitions occurred over approximately the same temperature-range as initial gelation on cooling (characterised by low-amplitude oscillatory measurements of G′ and G″) and increased in magnitude with increasing concentration of fructose. The displacement of both transitions, and particularly the exotherm, to progressively higher temperature as the rate of heating was increased was much greater than anticipated from simple thermal lag, indicating that the underlying structural changes are slow. The proposed interpretation is that fructose is capable of site-binding to pectin in both the ordered (threefold helix) and disordered state; the endotherm is attributed to helix melting and displacement of fructose; the subsequent exotherm is attributed to re-attachment of fructose to disordered chains, and the slow kinetics of this process to the conformational mobility of disordered pectin. On cooling over the same temperature range, a single exotherm was observed; the absence of detectable splitting is attributed to rapid re-attachment of fructose to conformationally-rigid helices. The magnitude of this endotherm (Δ H≈20 J/g) is close to the value found for cosolutes that show no evidence of site-binding, and to the net change in enthalpy for the endothermic and exothermic processes observed on heating, suggesting that the values of Δ H for displacement and re-attachment of fructose are essentially equal and opposite, with the net change coming from formation or melting of threefold helices. A smaller thermal process at higher temperature (endothermic on heating and exothermic on cooling) is attributed to hydrophobic association, which was also seen as an increase in G′ and G″ on heating in two consecutive cycles of temperature change.

Associative and segregative interactions between gelatin and low-methoxy pectin: Part 1. Associative interactions in the absence of Ca 2+

The effect of pH and mixing ratio on associative (electrostatic) interactions between gelatin (type B; pI=4.9) and low-methoxy pectin (DE 31.1) has been studied by differential scanning calorimetry (DSC), turbidity, and rheological measurements under low-amplitude oscillatory shear. All three techniques showed two thermal transitions for mixtures prepared below pH ∼3.5, one coincident with formation and melting of gelatin helices (below ∼35 °C) and the other centred at higher temperature (∼50 °C). The second transition was abolished by addition of salt (0.1 M NaCl), and is attributed to electrostatic association between the two polymers. Rheological measurements of network strength (storage modulus, G′) were consistent with conversion from a gelatin network augmented by additional crosslinking through pectin, to a (much weaker) pectic acid network augmented by additional crosslinking through gelatin as the relative proportion of pectin was increased. As observed in previous studies, compositions close to charge balance gave dense flocs on mixing. Mixtures prepared with either component in substantial (electrical) excess, however, remained homogeneous, suggesting solubilisation of the electrostatic complex by surplus charge. For these homogeneous preparations, the magnitude of the DSC endotherm corresponding to dissociation of the electrostatic complex remained roughly constant at ∼8 J/g of gelatin, and the magnitude of the first transition (below 35 °C) also remained roughly constant at a value close to that observed for gelatin alone (Δ H≈30 J/g). For compositions around charge balance, however, there was a massive increase in both endotherms, attributed to additional non-covalent interactions within flocculated particles. The lowest pH at which no significant electrostatic association could be detected by any of the investigative techniques was 3.9, which was therefore used in the study of segregative interactions in calcium pectinate–gelatin co-gels described in the following paper.

Iota-carrageenan/casein micelles interactions: evidence at different scales

The effect of temperature on the behaviour of iota-carrageenan (CI) 0.1 wt.%/casein micelles (CM) 0–5 wt.% mixtures has been studied using three techniques: confocal laser scanning microscopy (CLSM), differential scanning calorimetry (DSC) and spectrophotometry. The microscopy clearly shows that those mixed systems separate in two phases, one being enriched in CM. It has been shown that the CM concentration seems to have an effect on the extent of the phase separation phenomenon. The DSC experiments show that addition of CM modifies the helix to coil transition temperature of carrageenan. The enthalpy of melting of helices decreases as the CM concentration increases, and the peak is shifted towards higher temperature. Local electrostatic interactions between carrageenan chains and CM have been studied by a spectrophotometric method using methylene blue (MB) properties of absorption. The absorption spectra of MB in presence of CI and CM were compared with the one of MB in presence of carrageenan alone at temperatures above and below the carrageenan coil to helix transition. The modifications of the spectrum by addition of CM are discussed in terms of interactions and rigidification of the carrageenan chains.

A Study of Annealing and Freeze-Thaw Stability of Acid-Modified Tapioca Starches by Differential Scanning Calorimetry (DSC)

Tapioca starch was partially hydrolyzed by 6% (w/v) hydrochloric acid at room temperature for various lengths of time. Annealing and freeze-thaw stability of the acid-modified starches were studied using Differential Scanning Calorimetry (DSC). In the annealing study, as the hydrolysis time increased, the effect of annealing on narrowing and shifting the endothermic peak to a higher temperature was decreased. The endothermic transition of annealed 48-h acid-modified tapioca starch showed a narrow peak and a broad shoulder, corresponding to the melting of the amylopectin double helices (crystalline regions) and the retrograded partially hydrolyzed amylose, respectively. This effect of annealing on the sharpening of the endotherm was less pronounced on acid-modified tapioca starches annealed for 192 h and 768 h, respectively. These results indicated that annealing leads to more homogeneous crystallites and this effect is enhanced when the material contains more amorphous and homogeneous domains. In the case of the freeze-thaw stability study, the melting endotherm of recrystallized amylopectin became larger with increasing hydrolysis time. The first detectable endotherm of native tapioca retrograded gel was observed after five cycles, while all acid-modified retrograded gels showed the melting endotherm after only one cycle. Increasing hydrolysis time may increase the proportion of short chain amylose and amylopectin molecules, which are able to form double helices, resulting in an increase in the enthalpy and a higher retrogradation rate of the gel.

Structure and Chemical Properties of Partially Heated Corn Starch Granules

AbstractPartially heated (65°C) normal dent corn starch granules (5% aqueous suspensions) were fractionated according to granule size and apparent density. The fractions obtained were individually characterized by high performance size exclusion chromatography (HPSEC), differential scanning calorimetry (DSC), X‐ray diffraction, scanning electron microscopy and enzymatic susceptibility. Heat damaged granules were concentrated in a single fraction. The heat damaged fraction was highly susceptible to degradation by amyloglucosidase. HPSEC profiles of fractions showed differences in apparent solubility of amylose and amylopectin. The heat damaged fraction showed a high temperature (85°C) gelatinization endotherm in a DSC, indicating melting of double helices, but showed a V type X‐ray diffraction pattern. Starch granules exhibited different responses to heat because of inherent heterogeneity in their native state.

Melting behaviour of a triple helical polysaccharide schizophyllan in aqueous solution

Two sonicated samples of schizophyllan in aqueous solution at temperatures from 20 to 160°C were investigated by viscometry. The temperature dependence of the viscosity coefficient η showed that schizophyllan in water undergoes an irreversible thermal transition at about 135°C. The values of (ln η r) c ( ηr is the relative viscosity and c is the polymer concentration (w/v)) at 25°C determined after preheating aqueous schizophyllan indicated that the major conformations of schizophyllan in water at 120 and 150°C are triple helix and single random coil, respectively. Thus, it was concluded that the change in η at about 135°C with an increase in temperature is due to the melting of triple helices to single chains. Schizophyllan denatured to single chains at about 150°C did not restore the intact triple helix, but formed aggregates, when the solution was cooled to 25°C. It was also found that the aggregates form a gel when c is higher than a certain value.

Deoxydodecanucleotide heteroduplex d(TTTTATAATAAA). d(TTTATTATAAAA) containing the promoter Pribnow sequence TATAAT. I. Double-helix stability by UV spectrophotometry and calorimetry.

Thermally induced structural transition in the d(TTTTATAATAAA) d(TTTATTATAAAA) heteroduplex is characterized by UV-spectroscopy and differential scanning calorimetry. At low salt (less than 0.1 M) the occurrence of a cooperative transition in the lower temperature range, followed by a broad transition connected with small increase in absorbance is observed. At high salt (greater than or equal to 0.2 M) a single, monophasic transition appears. Linear dependence of the latter on log of salt concentration (dTm:dlogM = 14.2 degrees C) and of 1/Tm on log of oligomer concentration [derived therefrom delta H (v.H.) = 77.1 kcal/mole (duplex)] allows relating it to the melting of the heteroduplex helix. The non-cooperative transition, independent of oligomer concentration and similar to that of the single chain, was attributed to melting of short hairpin helices upon heteroduplex dissociation. Calorimetric enthalpy: 75.6 kcal/mole (duplex) proved significantly lower than predicted from known calorimetric data for poly[d(AT)] and poly d(A) X poly d(T).

X‐ray studies of smectic‐crystal and crystal‐smectic transition in oriented polypropylene

AbstractThe results of the study of polypropylene crystallization from the oriented paracrystalline state and the transition to the smectic state observed during isometric melting of the samples crystallized in this manner are interpreted from the viewpoint of structural transition in helical polypropylene macromolecules inevitably involving the local melting of helices.

Poly(U)-directed peptide-bond formation from the 2'(3')-glycyl esters of adenosine derivatives.

The self-condensation of 2'(3')-O-glycyl esters of adenosine, adenosine-5'-(O-methylphosphate) and P1, P2-diadenosine-5'-pyrophosphate in 6.2 mM solutions at pH 8.0 and -5 degrees C in the presence of 12.5 mM poly(U) yields approximately 3 times as much diketopiperazine as reactions without poly(U). As the concentration of 2'(3')-O-(glycyl)-P1, P2-diadenosine-5'-pyrophosphate is decreased from 6.2 mM to 1.5 mM the yield of diketopiperazine in the presence of poly(U) decreases slightly from 6.6% to 5.2%, whereas, in the absence of poly(U) the yield of diketopiperazine decreases substantially from 2.4% to 0.75%. The enhanced yield of diketopiperazine that is attributed to the template action of poly(U) is temperature dependent and is observed only at temperatures below 10 degrees C (5 degrees C to -5 degrees C) for 6.2 mM 2'(3')-O-(glycyl)-adenosine-5'-(O-methylphosphate) and below 23 degrees C (15 degrees C to -5 degrees C) for 6.2 mM 2'(3')-O-(glycyl)-P1, P2-diadenosine-5'-pyrophosphate. The absence of a template effect at high temperatures is attributed to the melting of the organized helices. The hydrolysis half-lives at pH 8.0 and -5 degrees C of 2'(3')-O-(glycyl)-adenosine, 2'(3')-O-(glycyl)-adenosine-5'-(O-methylphosphate), 2'(3')-O-(glycyl)-P1, P2-diadenosine-5'-pyrophosphate, and 5'-O-(glycyl)-adenosine in the presence of poly(U) are substantially larger than their half-lives in the absence of poly(U). The condensation of 2'(3')-O-(glycyl)-adenosine yields 5% of 5'-O-(glycyl)-adenosine in the presence of poly(U) compared to 0.7% in the absence of poly(U).

The molecular mechanism of thermal unfolding of Escherichia coli formylmethionine transfer RNA

The molecular mechanism of thermal unfolding of Escherichia coli tRNA fMet (in 0.17 m-NaCl without Mg 2+) has been elucidated by a combination of relaxation kinetics and proton nuclear magnetic resonance spectroscopy. We measured the n.m.r. ‡ ‡ n.m.r., nuclear magnetic resonance; hUra, dihydrouracil. spectrum of the hydrogen-bonded ring NH protons at different temperatures and found that the resonances assigned to each arm of the cloverleaf broaden and disappear together, yielding four distinct n.m.r. “melting” transitions. Temperature-jump measurements in the same solvent showed five co-operative melting transitions, varying in relaxation time from a few microseconds to ten milliseconds. The relaxation and n.m.r. measurements were correlated by the following model. When the lifetime of a hydrogen-bonded proton in a helix is five milliseconds, its n.m.r. line will be broadened to approximately twice its intrinsic low-temperature width and appear to “melt”. The helix dissociation time constants of the relaxation effects were extrapolated by the Arrhenius equation to lower temperatures where their values were five milliseconds. The correlation of extrapolated dissociation time constants with n.m.r. melting of specific helices allowed assignments of the structural basis for each relaxation effect. The results show that the principal path for the reversible thermal unfolding of tRNA 1 fMet under these solution conditions is first, transient opening of the dihydrouridine helix, followed by simultaneous melting of the dihydrouridine helix and a “tertiary” interaction, which does not correspond to a cloverleaf helix. The tertiary interaction is much less stable in tRNA 3 fMet, with T m lowered by 16 °C from tRNA 1 fMet. The sequence of melting steps at higher temperatures is the same in the two isoacceptors: first the TΨC helix melts, followed by the anticodon helix and finally the acceptor stem helix. Thermodynamic and kinetic parameters are reported for these steps. The method of sequential melting, combining n.m.r. and relaxation kinetic techniques, is a powerful procedure for elucidating RNA secondary structure. In addition, this method allows assignment of many hydrogen-bonded ring NH proton resonances that are unresolved in the low-temperature spectrum.