Heats of the helix-coil transitions of the poly A-poly U complexes

The heats of the conformational transitions of poly (A + U) and poly(A + 2U) were determined in a twin-cell differential thermal analysis microcalorimeter capable of measuring heat effects of 25–35 meal, in reactions of this type, with a precision of about 3%. The dependence of these heats on the concentration of Na⁺ and K⁺ was studied and was found to be interpretable with fair success by a simple phenomenological argument which employs the concept of binding of counterions to polyelectrolytes.     

DNA structural transitions induced by divalent metal ions in aqueous solutions

Using methods of IR spectroscopy, light scattering, gel-electrophoresis DNA structural transitions are studied under the action of Cu2+, Zn2+, Mn2+, Ca2+ and Mg2+ ions in aqueous solution. Cu2+, Zn2+, Mn2+ and Ca2+ ions bind both to DNA phosphate groups and bases while Mg2+ ions-only to phosphate groups of DNA. Upon interaction with divalent metal ions studied (except for Mg2+ ions) DNA undergoes structural transition into a compact form. DNA compaction is characterized by a drastic decrease in the volume occupied by DNA molecules with reversible formation of DNA dense particles of well-defined finite size and ordered morphology. The DNA secondary structure in condensed particles corresponds to the B-form family. The mechanism of DNA compaction under Mt2+ ion action is not dominated by electrostatics. The effectiveness of the divalent metal ions studied to induce DNA compaction correlates with the affinity of these ions for DNA nucleic bases: Cu2+>Zn2+>Mn2+>Ca2+>Mg2+. Mt2+ ion interaction with DNA bases (or Mt2+ chelation with a base and an oxygen of a phosphate group) may be responsible for DNA compaction. Mt2+ ion interaction with DNA bases can destabilize DNA causing bends and reducing its persistent length that will facilitate DNA compaction.     

Competing B-Z and helix-coil conformational transitions in supercoiled plasmid DNA.

The formation of melted regions from A + T-rich sequences and left-handed Z-DNA by alternating purine-pyrimidine sequences will both be facilitated by negative supercoiling, and thus if the sequences are present within the same plasmid molecule they will compete for the free energy of supercoiling. We have studied a series of plasmids that contain either (CG)8 or (TG)12 sequences in either G + C or A + T-rich contexts, by means of two-dimensional gel electrophoresis and chemical modification. We observe both B-Z and helix-coil transitions in all plasmids at elevated temperatures and low ionic strength. The plasmids fall into a number of different classes, in terms of the conformational behavior. As the superhelix density is increased, pCG8/vec ((CG)8 in G + C-rich context) undergoes an initial B-Z transition, followed by melting transitions in sequences remote from the (CG)8 sequence. The two transitions are coupled through the topology of the molecule but are otherwise independent. When the (CG)8 sequence was placed in an A + T-rich context (pCG8/col), the helix-coil transition was perturbed by the presence of the Z-DNA segment. Replacement of the (CG)8 tracts by (TG)12 sequences resulted in a further level of interaction between the transitions. Statistical mechanical modeling of the transitions suggested that at intermediate levels of negative supercoiling the Z-DNA formed by the (TG)12 sequence has a lowered probability due to the helix-coil transition in the A + T-rich sequences. These studies illustrate the complexities of competing conformational equilibria in supercoiled DNA molecules.     

Enthalpy of helix-coil transitions from heats of solution. I. Polyglutamates

The heat of solution of a series of three polyglutamates as a function of solvent composition was measured. The abrupt increase in heat of solution at the solvent composition of the helix–coil transition (as evidenced by optical rotation data) allows the estimation of the transition enthalpy change. The difference of side chain in the three polyglutamates has no appreciable effect on the transition enthalpy, although it affects the helix stability, as judged from the solvent composition at the transition points. These facts are discussed on the basis of existing models of the transition.     

Conformational transitions of schizophyllan in aqueous alkaline solution

The conformational transitions of schizophyllan were studied in aqueous alkaline solutions by high-sensitivity differential scanning calorimetry (DSC) and optical rotation measurements. The temperature of half completion for reversible intramolecular conformational transition determined by DSC, centered at 7.4°C in water, increases to 37.2°C at 0.01M KOH with increasing alkaline concentration. The transition enthalpy per mole of the polysaccharide repeating unit is 2.62 ± 0.23 kJ mol⁻¹ independent of the alkaline concentration. The cooperative unit size for the transition decreases with increasing alkaline concentration. Optical rotation was measured as a function of pH at 25 and 60°C. A sharp decrease in optical rotation was observed at pH = 13, which is ascribed to the triple helix-coil transition. From data obtained by DSC and optical rotation measurements, in combination with results reported previously, a phase diagram for the conformation of schizophyllan as a function of temperature and pH is proposed. The irreversibility of the triple helix to single coil transition, induced by strong alkali, was investigated as a function of polymer concentration by gel permeation chromatography and electron microscopy. The renatured samples at polymer concentrations < 1.0 mg/mL, which are prepared by dissolution in 0.25M KOH followed by neutralization with HCl, are observed as a mixture of globular, linear, and circular structures, and larger aggregates with less-defined morphology by electron microscopy. Higher concentrations lead to increased proportions of multichain clusters (aggregates). Subsequent annealing of the renatured samples at 115–120°C increases the proportion of circular species. The change in molecular weight distribution of samples that accompanies the renaturation and annealing mentioned above can be well interpreted in terms of the proportion of species having different morphology as observed by electron microscopy. © 1996 John Wiley & Sons, Inc.     

Differential detergent solubility investigation of thermally induced transitions in cytochrome c oxidase

The thermal denaturation of membrane-reconstituted cytochrome c oxidase (EC 1.9.3.1) occurs at approximately 63 degrees C as determined by high-sensitivity differential scanning calorimetry. The heat capacity profile associated with this process is characterized by the presence of two well-defined peaks, indicating that all the enzyme subunits do not have the same thermal stability. This thermal denaturation of the enzyme complex is coupled to a change in its solubility properties. This change in solubility allows separation of the native and denatured protein fractions by detergent solubilization followed by centrifugation under conditions in which only the native fraction is solubilized. Using this principle, it has been possible to study the denaturation of membrane-reconstituted cytochrome c oxidase and quantitatively identify the protein subunits undergoing thermal denaturation using computer-assisted gel electrophoresis analysis. This technique allows calculation of single-subunit thermal denaturation profiles within the intact enzyme complex, and as such, it can be used to obtain transition temperatures, molecular populations, and van't Hoff enthalpy changes for individual protein subunits, thus complementing results obtained by high-sensitivity differential scanning calorimetry.     

Enthalpy of helix-coil transitions from heats of solution. II. Poly-epsilon-carbobenzoxy-L-lysine and copolymers with L-phenylalanine

The heal of solution of Poly-ε-carbobenzoxy-L -lysine and of a series of its copolymers with phenylalanine was measured as a function of solvent composition. The enthalpy change of the helix-to-coil transition was estimated for the various cases. The previous findings that side chains do not greatly affect the transition enthalpy is confirmed also in cases having much larger differences in helix stability.     

Molecular field theory of hysteresis in helix-coil transitions of polynucleotides

A molecular field theory, taking into account long-range electrostatic forces is used to study helix–coil transitions of polynucleotides. The theory predicts the existence of hysteresis when the electrostatic interaction parameter is large compared to the thermal energy. The theory is applied to the acid–base titration of poly(A)·2 poly(U).     

DNA helix-coil transition in alkaline medium

Dna helix-coil transition in th alkaline medium was considered theoretically and experimentally. On the basis of the theory and experimental comparison the DNA double-stranded form deprotonation was revealed.     

Comparison of several calculations of helix-coil transitions in heterogeneous polymers

Random number methods are used to calculate helix—coil transition curves for the model of a heterogeneous polypeptide of random sequence. These curves are compared with several other calculations. The random number computations confirm the exact calculation of Lehman; among the several approximate calculations examined only that of Fixman and Zeroka agrees closely with results of the random number method over the whole range of conditions considered. Calculations are also reported of the average length of helix and coil sections in a heterogeneous molecule of random sequence which is undergoing the helix-coil transition.     

Theoretical study of volume changes associated with the helix-coil transition of an alanine-rich peptide in aqueous solution

The changes in the partial molar volume (PMV) associated with the conformational transition of an alanine-rich peptide AK16 from the alpha-helix structure to various random coil structures are calculated by the three-dimensional interaction site model (3D-RISM) theory coupled with the Kirkwood-Buff theory. The volume change is analyzed by decomposing it into contributions from geometry and hydration: the changes in the van der Waals, void, thermal, and interaction volume. The total change in the PMV is positive. This is primarily due to the growth of void space within the peptide, which is canceled in part by the volume reduction resulting from the increase in the electrostatic interaction between the peptide and water molecules. The changes in the void and thermal volume of the coil structures are widely distributed and tend to compensate each other. Additionally, the relations between the hydration volume components and the surface properties are investigated. We categorize coil structures into extended coils with the PMV smaller than helix and general coils with the PMV larger than helix. The pressure therefore can both stabilize and destabilize the coil structures. The latter seems to be a more proper model of random coil structures of the peptide.     

Microwave dielectric absorption of DNA in aqueous solution

The dielectric properties of aqueous solutions of DNA were measured at frequencies ranging from 0.1 to 12 GHz. The results are analyzed using the Maxwell mixture theory and yield a value for the hydration of the DNA of about 0.4 g/g, which is in the range observed in other investigations. No evidence was found for an additional absorption effect at microwave frequencies, which has been predicted to occur in certain DNA analogs due to the vibrational excitation of the double helix by the applied microwave field.     

Kinetics of the helix-coil transition in DNA

The kinetics of the helix-coil transition have been investigated for T2 and T7 phage DNA in a formamide-water-salt mixed solvent using a slow temperature perturbation technique (applicable to kinetic processes with rate constants ? 3 min^?1). In this solvent degradation of the DNA is effectively suppressed. Complex kinetic curves are observed by absorbance and viscosity measurements for the response to denaturing perturbations in the transition region. Analysis of the decay curves indicates that the denaturation reaction in this time range can be treated as a first-order reaction with a variable first-order rate parameter, k, the derivative of the logarithm of the absorbance or viscosity change with respect to time. In the approach to denaturation equilibrium in the transition region, the rate parameter is determined only by the instantaneous extent of denaturation of the molecules. Near equilibrium, the rate parameter assumes a constant value characteristic of the equilibrium state. In this region, where the denaturation reaction proceeds as a simple first-order process, both the decay of absorbance (reflected local conformational change) and the decay of solution viscosity (reflecting macromolecular conformational change) are characterized by the same constant value of k. In 83% formamide, 0.3M Na⁺, the rate parameter k for T2 DNA decreases from an extrapolated value of 2.0 min^?1 at 0% denaturation to 0.11 min^?1 at 90% denaturation. Rate parameters determined for T7 DNA at the same counterion concentration and fraction of denaturation are approximately five times as large as those cited for T2 DNA, indicating an inverse proportionality of rate constant to molecular length. On the other hand, simple first-order kinetic responses with constant k are obtained for renaturing perturbations within the transition, indicating that the mechanism of rewinding differs, in most cases, from that of unwinding. Only in the limit of very small perturbations about a given equilibrium position are the rate constants k obtained from denaturing and renaturing perturbations equal. For perturbations of finite size, it appears possible that an intramolecular initiation or nucleation event may precede rewinding and limit the rate of this reaction. The rate parameters again are approximately inversely proportional to molecular weight. The one exception to the first-power dependence on molecular weight appears when temperature jumps are made upward into the post-transition region. Here the molecular-weight dependence is second power, but complications arising from the different strand-separation properties of T2 and T7 DNA's make interpretation difficult. The previously used model of friction-limited unwinding appears to fit all the observations except for the molecular-weight dependence.     

Thermal transitions of DMPG bilayers in aqueous solution: SAXS structural studies

Dimyristoylphosphatidylglycerol (DMPG) has been extensively studied as a model for biological membranes, since phosphatidylglycerol is the most abundant anionic phospholipid in prokaryotic cells. At low ionic strengths, this lipid presents a peculiar thermal behavior, with two sharp changes in the light scattering profile, at temperatures named here T(on)(m) and T(off)(m). Structural changes involved in the DMPG thermal transitions are here investigated by small angle X-ray scattering (SAXS), and compared to the results yielded by differential scanning calorimetry (DSC) and electron spin resonance (ESR). The SAXS results show a broad peak, indicating that DMPG is organized in single bilayers, for the range of temperature studied (10-45 degrees C). SAXS intensity shows an unusual effect, starting to decrease at T(on)(m), and presenting a sharp increase at T(off)(m). The bilayer electron density profiles, obtained from modeling the SAXS curves, show a gradual decrease in electron density contrast (attributed to separation between charged head groups) and in bilayer thickness between T(on)(m) and T(off)(m). Results yielded by SAXS, DSC and ESR indicate that a chain melting process starts at T(on)(m), but a complete fluid phase exists only for temperatures above T(off)(m), with structural changes occurring at the bilayer level in the intermediate region.     

Mechanically induced helix-coil transition in biopolymer networks

The quasi-equilibrium evolution of the helical fraction occurring in a biopolymer network (gelatin gel) under an applied stress has been investigated by observing modulation in its optical activity. Its variation with the imposed chain extension is distinctly nonmonotonic and corresponds to the transition of initially coiled strands to induced left-handed helices. The experimental results are in qualitative agreement with theoretical predictions of helices induced on chain extension. This new effect of mechanically stimulated helix-coil transition has been studied further as a function of the elastic properties of the polymer network: crosslink density and network aging.     

Electrostatic forces at helix-coil boundaries in DNA

The Tm of internal loop-forming (dA.dT)N domains in pBR322 DNA has been measured over a tenfold range of [Na+]. The slopes SN = dTm/d log [Na+] are linear and decrease in magnitude with decreasing loop size N, signaling a reduction in Na+ released during the transition of these domains to the coil state. Values of SN decrease linearly with increasing N-1 in accordance with the expectation of a simple model for the occurrence of a gradient of long-range electrostatic forces at helix-coil boundaries, and extrapolate almost precisely to the value of S infinity observed for (dA.dT) infinity. These results indicate (1) less counterion is released per phosphate residue from the finite loop than from the infinite-sized loop, and (2) the difference in binding is constant for each boundary formed and independent of the size of the loop within the range examined: approximately 350 base pair (bp) greater than N greater than 71 bp. The slope of the dependence of SN on N-1 indicates the region of higher charge density at the boundary extends at least 18 A into the coil and probably 40-50 A before dropping to a value characteristic of the unperturbed coil. The free energy for excess counterion binding at boundaries can be expressed by -delta G/RT = 10.47 log[Na+] + 5.234 When the loop entropy function in a statistical mechanical algorithm for the dissociation of DNA is weighted by this quantity, calculated Tm are seen to vary by only +/- 0.09 degrees C from observed.     

The kinetics of DNA helix-coil subtransitions

The kinetic analysis of individual helix-coil subtransitions were performed by comparing melting and renaturation profiles obtained at different temperature change rates. The duration of the three transition stages and its dependence on temperature and ionic strength were determined for a T7 phage DNA fragment. The obtained temperature dependence of the melting time for a stretch flanked by melted regions is in quantitative agreement with that predicted by the theory of slow processes (V.V. Anshelevich, A.V. Vologodskii, A.V. Lukashin, M.D. Frank-Kamenetskii, Biopolymers 23, 39 (1984)). The reasons are discussed for the increasing relaxation time of this stretch in the middle of its transition with decreasing ionic strength. The zipping kinetics of a melted region under essentially nonequilibrium conditions was examined for T7 fragment and pAO3 DNAs. The obtained temperature dependence of the zipping time is in quantitative agreement with calculations based on the theory of slow processes. The renaturation times of stretches flanked by helical regions proved fairly small even at a low ionic strength. These times are several orders of magnitude smaller than the renaturation times of the same stretches with one helical boundary. A formal application of the theory of slow processes failed to account for the small renaturation times of stretches that are zipped from both ends. This is probably due to the non-allowance for the changing entropy of the loop linking two helix-coil boundaries migrating towards each other. Slow processes have been revealed in the intramolecular melting of Col E1 DNA at a high ionic strength. The reason for the long relaxation time of one subtransition is the large size of the loop that separates the melting stretch from the helical part of the molecule. This result can be accounted for by the theory of slow processes.