Melting of DNA in ethanol–water solutions

The thermal denaturation curves of DNA have been investigated over a wide range of temperatures T and ethanol concentrations C by CD and uv absorption methods. The phase diagram of conformational DNA states has been constructed in T,C plane. The range of the A, B, and coiled forms of DNA was determined. The behavior of the DNA denaturation curve in the neighborhood of the triple point shows that the conformational transition B → A is realized by short parts of the DNA double helix. This is the reason for the coincidence of the melting temperatures of the A and B forms of DNA throughout the range of their coexistence.     

Mechanochemical study of NaDNA and NaDNA-netropsin fibers in ethanol-water and trifluoroethanol-water solutions.

Highly oriented calf-thymus NaDNA fibers, prepared by a wet-spinning method, were complexed with netropsin in ethanol-water and trifluoroethanol (TFE)-water solutions. The relative fiber length, L/L0, was measured at room temperature as a function of ethanol or TFE concentration to obtain information on the B-A conformational transition. The B-A transition point and transition cooperativity of the fibers were calculated. The binding of netropsin to NaDNA fibers was found to stabilize B form and to displace the B-A transition to higher ethanol concentration, as indicated by its elongational effect on the fiber bundles. An increased salt concentration was found to reduce netropsin binding. In netropsin-free ethanol solution, the dissociation of bound netropsin from the DNA fibers was observable. Pure B-NaDNA fibers were found to be more stable in TFE solution than in ethanol solution. This was interpreted as being due to a different steric factor and a larger polarity of TFE compared with ethanol, resulting in its smaller capacity to reduce the water activity and dielectric constant of the medium in the immediate vicinity of DNA fibers. Therefore, the effect of netropsin binding on the B-A transition of NaDNA fibers became less obvious in TFE solution. In another series of experiments, L/L0 was measured as a function of temperature to obtain information on the helix-coil transition, or melting, as well as the B-A transition of NaDNA and NaDNA-netropsin fibers. The melting temperature and helix-coil transition width were calculated from the melting curves. A phenomenological approach was used to describe the melting behavior of the fibers in and around the B-A transition region. The effect of netropsin on the melting of DNA fibers was attributed mainly to the stabilization of B-DNA and to a higher melting cooperativity in the B-DNA region.     

Theoretical study of large conformational transitions in DNA: the B<-->A conformational change in water and ethanol/water

We explore here the possibility of determining theoretically the free energy change associated with large conformational transitions in DNA, like the solvent-induced BA conformational change. We find that a combination of targeted molecular dynamics (tMD) and the weighted histogram analysis method (WHAM) can be used to trace this transition in both water and ethanol/water mixture. The pathway of the transition in the A→B direction mirrors the B→A pathway, and is dominated by two processes that occur somewhat independently: local changes in sugar puckering and global rearrangements (particularly twist and roll) in the structure. The B→A transition is found to be a quasi-harmonic process, which follows closely the first spontaneous deformation mode of B-DNA, showing that a physiologically-relevant deformation is in coded in the flexibility pattern of DNA.     

A method for the experimental study of DNA conformational transitions in fibers

The method proposed for the study of DNA conformational transitions is based on the proportionality, experimentally observed, between the length of a DNA fiber and the axial rise per nucleotide characterizing the molecular helix. Precise curves for the A-B and B-C transitions as a function of the relative humidity are obtained by using X-ray fiber data and measurements of fiber dimensions. It is thus shown that the A-B transition is a cooperative process between two different states, whereas the B-C transition can be considered as a progressive change of conformation. The present method is applied on two natural DNAs differing in base composition so that the effect of the nucleotide content on the conformational changes can be estimated.     

Effect of binding on melting transitions in polymer–diluent systems

An expression is derived for the melting point of a polymer when in equilibrium with a solution in which binding of low molecular weight compounds to the polymer takes place. Allowance is made for the possibility that the crystalline polymer itself is a complex. The argument is a purely thermodynamic one and is based on a consideration of the change in free energy as a result of a change in binding. Allowance is made also for non-specific polymer–solvent interactions, in which the mixture of low molecular weight solvents is treated as a single solvent. Special attention is paid to “inverted” melting transitions, i.e., cases in which the melting point increases with increasing dilution of the polymer. It is shown that as a rule this is accompanied by a corresponding, inverted effect of the solvent composition on the melting point. It is further shown that-in the absence of binding, “normal” behavior at the critical point (i.e., phase separation is induced by lowering the temperature) is always accompanied by “normal” melting behavior (i.e., a decrease in melting point when the polymer is diluted). Also, “inverted” melting always implies that phase separation at the critical point is induced by heating, but the reverse is not necessarily true.     

Helix–random chain transitions of polyamino acids in organic acid–salt solutions

Polyamino acids which are soluble and helical in acetic acid and dichloroacetic acid (DCA) have been observed to undergo a helix to random chain transition upon the addition of lithium salts of strong acids. The transition can be reversed by diluting the salt. Apparently only lithium cations are able to bring about the polycarbobenzoxy-L -lysine (PCBL) transition in acetic acid, whereas the anions display a varying degree of effectiveness; ClO₄^? > Br^? > TSA^? > Cl^? > NO₃^?. The lithium salts of carboxylate anions such as OAc^? and TFA^? do not cause polymer unwinding in acetic acid. Neither do the acids, TSA, HCl, TFA, or DCA induce the transformation in acetic acid. Poly-L -alanine (PLA) in DCA unfolds as LiBr is added, but does not unfold in the presence of 0.5M (CH₃)₄NBr, 0.25M CsBr, or 0.32M HCl. These results are explained on the basis of a direct interaction of the lithium salt with the polymer amide groups to form an ion-pair complex. The extent to which the union of the ion pair can dissociate from the complex in the low dielectric constant, environment determines the degree of unfolding of the polymer. The anion dissociation equilibrium presumably therefore would lie in the same order as given above. Acids such as HCl and TSA are considered to substantially protonate and ion-pair with the polymer, but do not readily dissociate the anion partner from the complex, and therefore do not produce an unstable positively charged helical structure.     

Phase transitions of the starch–water system

When purified potato starch granules are heated in the presence of limited amounts of water (less than 1.5 H₂O: starch, w/w), two endothermic transitions are observed by differential scanning calorimetry. The lower temperature endotherm is always observed at a fixed temperature, 66°C; it is the only endotherm observed when excess water is present. The higher temperature endotherm is observed at increasing temperatures as the water content is decreased. The size of this endotherm decreases with water content. The appearance of the higher temperature endotherm allows the determination of the stoichiometry for full hydration of starch, 14 H₂O/hexose unit. The shift of the higher temperature endotherm is interpreted as the lowering of the melting point of starch crystallites by solvent water.     

Coupling between the helix–coil transition and nematic–isotropic transitions in polypeptide solutions

The lattice model of Flory has been extended in order to consider equilibrium between isotropic and nematic phases containing helix–coil type chains. Nearly complete exclusion of coil sequences from the lyotropic nematic phase produces an enhanced cooperativity in the helix–coil transition. In poor solvents this enhancement begins to occur at concentrations typical of some experiments.     

The application of surface tension measurements to the study of conformational transitions in aqueous solutions of poly-L-lysine

The change in surface tension of solutions of poly-L -lysine in water has been studied as a function of temperature at various pH values. The changes at various temperatures have been correlated with changes in the circular dichroic spectra reflecting conformational change. In addition to the major transition at 50°C attributed to the conversion of the α-helical → β conformation, two other transitions have been observed at 30°C and 80°C. A minimum in the surface tension value was observed at pH 10, near the pK value for poly-L -lysine. It was concluded that at this pH the concentration of hydrophobic groups at the surface was a maximum.     

A differential scanning calorimetric study of the conformational transitions of schizophyllan in mixtures of water and dimethylsulfoxide

The thermal conformational transitions of two sonicated samples of schizophyllan were studied in water-dimethylsulfoxide (DMSO) mixtures by high-sensitivity differential scanning calorimetry (DSC). Two transitions were observed over most of the range of solvent compositions. These were assigned to an internal change of the triple helix [T. Itou et al. (1986) Macromolecules 19, 1234-1240] and a triple-helix-single-coil transition [T. Sato et al. (1981) Carbohydr. Res. 95, 195-204], respectively. In water, the former transition observed at lower temperature for a low molecular weight sample, U-1, is centered at 3 degrees C and characterized by the specific enthalpy, delta hcal = 3.29 J g-1. A higher molecular weight sample, M-2, showed this transition at 7 degrees C with delta hcal = 4.39 J g-1. The transition temperature for both samples increased with increasing DMSO concentration up to about 50 degrees C at 70 weight % DMSO, and then rapidly decreased with increasing DMSO concentration, with about 3 degrees C higher for M-2 than for U-1 over the DMSO concentration. The transition was not observed when the concentration of DMSO exceeded 87%. It was found that delta hcal for both samples was a linear function of t 1/2, the temperature of half-completion in degrees C, delta hcal = 0.177t + 2.96. The triple helix-coil transition was observed at around 127 degrees C for U-1 and above 130 degrees C for M-2 in the range of DMSO composition below about 70%. The transition temperature decreased with increasing DMSO concentration at above 70%, and the transition finally disappeared when the DMSO concentration exceeded 90%. The plot of delta hcal vs. t 1/2 for the transition of both samples gave a linear relation, delta hcal = 0.253t - 10.58. The reversibility of the transition at lower temperature was demonstrated by the reversibility of the curves when the first heating was stopped before the second transition. Once the heating was performed over the second transition, the reheating DSC curves showed several endothermic peaks, indicating the irreversibility of the transition and heterogeneity in the conformation of the heated schizophyllan.     

Multiple conformational transitions of hen egg-white lysozyme in aqueous acetic acid solutions

Circular dichroism measurements revealed that hen egg-white lysozyme underwent multiple conformational transitions upon the addition of acetic acid. The transitions were reversible as judged from complete recoveries of enzymatic activity, electrophoretic mobility in SDS-polyacrylamide gel, and of ellipticity. Two transitions, with the mid-concentrations of 26 and 38% (v/v), were observed with the CD spectra in the amide absorption region. The two transitions were essentially athermal in the temperature ranges, 0 to 25 degrees C for the former and -10 to 10 degrees C for the latter. The trough ellipticity for the product of the transition at the higher acetic acid concentration (DII form) very closely approached the value for the synthetic polypeptides in the beta-conformation as the temperature was lowered. Molecular weight measurements by sedimentation equilibrium indicated that the products were both monomeric. Measurements of CD spectra in the aromatic absorption region showed another transition, whose mid-concentration varied with temperature from 26% (v/v) (at about 25 degrees C) to 38% (v/v) (at -10 degrees C). A change in the hydrodynamic volume detectable by exclusion chromatography was associated with this transition only.     

Effect of divalent metal ions on DNA conformational transitions in water-ethanol solutions

The influence of different MgCl2 and MnCl2 concentrations on DNA conformational transitions in water-ethanol solutions was studied. It was shown that the presence of magnesium ions in solution at a concentration of 5 x 10(-4) M did not influence the decrease in the size of DNA without change in its persistent length at an alcohol concentration of about 17 % v/v. In contrast, manganese ions prevent this change in DNA parameters. At sufficiently high ethanol concentrations, the compaction of DNA followed by its precipitation takes place, which is accompanied by an increase of scattering in solution. As the concentration of Mg2+ and Mn2+ in solution increases, this process is observed at lower ethanol concentrations.     

Thermal denaturation of Na- and Li-DNA in salt-free solutions

Thermal denaturation of Na- and Li-DNA from chicken erythrocytes was studied by means of scanning microcalorimetry in salt-free solutions at DNA concentrations (C_p) from 4.5 · 10^?2 to 1 · 10^?3 moles of nucleotides/liter (M). Linear dependencies of DNA melting temperature (T_m) vs lgC_p were obtained: ((1)) ((2)) for Na- and Li-DNA, respectively. Microcalorimetry data were compared with the results of spectrophotometric studies at 260 nm of DNA thermal denaturation in Me-DNA + MeCl solutions at C_p ? (6–8) · 10^?5 M and C_s = 0–40 mM (Me is Na or Li, C_s is salt concentration). It was found that Eqs. (1) and (2) are valid in DNA salt-free solutions over the C_p range 6 · 10^?5?4.5 · 10^?2M. Protonation of DNA bases due to the absorption of CO₂ from air in Na-DNA + NaCl solutions affects DNA melting parameters at C_s < 4 mM. Linear dependence of T_m on lga₊ is found in Na-DNA + NaCl at C_s > 0.4 mMin the absence of contact of solutions with CO₂ from air (a₊ is cation activity). A dependence of [dT_m/dlga₊] on Li⁺ activity was observed in Li-DNA + LiCl solutions at C_s < 10 mM: [dT_m/dlga₊] increases from 17°–18° at C_s > 10 mM to 28°–30° at C_s ? 0.2–0.4 mM. Spectrophotometric measurements at 282 nm show that this effect was caused by protonation of bases in fragments of denatured DNA in neutral solutions. The Poisson–Boltzmann (PB) equation was solved for salt-free DNA at the melting point. The linear dependence of T_m vs lgC_p was interpreted in terms of Manning's condensation theory. PB and Manning's theories fit the experimental data if charge density parameter (ξ) of denatured DNA is in the range 1.8–2.1 (assuming for native DNA ξ = 4.2). Specificity of Li ions in interactions with DNA is discussed. © 1994 John Wiley & Sons, Inc.     

Fluorescence study of conformational transitions in the structure of myoglobin

Fluorescence studies of myoglobin and Mb-like structures, apomyoglobin and the complex of apo-Mb with protoporphyrin IX, reveal both the similarity between them, which is due to a common type of polypeptide chain folding, and the distinctions imposed by the influence of the prosthetic group. Close resemblance of structures of holomyoglobin and its metal-free analog, PPIX--apo-Mb, points to a key role of specific interactions between the protein and the protoporphyrin macrocycle rather than the Fe-protein bond in the formation of Mb-like structures. In PPIX--apo-Mb, both the hydrophobic core and the important ionic bonds between different structural elements () stabilizing the Mb structure are almost completely retained. The bond between Fe and proximal His-F8 allows additional integration of the structures of the heme cavity and the myoglobin molecule as a whole, providing its functional activity and highly cooperative conformational transitions. In all the myoglobin-like structures studied, a certain relationship is found between conformational states of the , the heme cavity, and the N-terminal part of the molecule. This is probably due to variations in the mutual orientation of the ABCDE and FGH helical domains, depending on the interactions between the protein, the prosthetic group, and the ligand in the heme crevice. The correlation between conformations of the N-terminal and heme regions found at a level of the globin tertiary structure is very important for understanding the mechanisms of homo- and heterotropic regulation in tetrameric hemoglobins.     

A study of DNA melting in concentrated water-alcohol solutions

The DNA melting profiles with high resolution have been studied for conditions corresponding to the B and A conformations of DNA in water-alcohol solutions. The melting profiles of the A-form and B-form DNA, their mean melting temperatures and melting range width were found to differ. DNA was shown to be heterogeneous in respect of the B-A transition, the GC-rich regions more readily converting into the A form than AT-rich ones. The presence of boundaries between the A and B sections within the transition zone did not smooth off the fine structure of melting profiles.     

Comparative study of the conformational transitions of frog and rabbit phosphorylases B

1. Conformational motility of the purified muscle glycogen phosphorylase B from two species of vertebrates (rabbit and frog) was investigated by the Hydrogen-Exchange method and Infrared Spectometry. 2. The experimental results of the 1H-2H exchange were expressed in terms of the probability P of exposure to isotopic solvent of phosphorylase peptide groups and in terms of the corresponding changes in standard free energy delta Go. 3. The combined methods used didn't show considerable differences of the protein conformations in the physiological pH region but rabbit phosphorylase was only characterized by rather more compact structure in comparison with frog phosphorylase.     

An FT-IR study of DNA and RNA conformational transitions at low temperatures

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO2 and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant. Marker infrared bands for the B conformer have been found to be the strong band at 825 cm-1 (sugar conformer mode) and a band with medium intensity at 690 cm-1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm-1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm-1 and at 665-600 cm-1.