Acceleration of the refolding of Arc repressor by nucleic acids and other polyanions.

The refolding rate of the Arc repressor dimer can be accelerated 30-fold or more by negatively charged polymers including single-stranded and double-stranded DNA, RNA, and polyvinylsulfate but not by neutral or positively charged polymers. The salt-dependence of the polyanion-mediated process and mutant studies indicate that electrostatic interactions are important in the rate acceleration. Urea-dependence studies suggest that Arc is relatively unstructured in the transition state for polyanion-stimulated refolding. At low ionic strength, the observed kinetics of refolding are consistent with a model in which denatured Arc monomers bind rapidly and nonspecifically to the polyanion and complete folding in the bound state.

Formation of a denatured dimer limits the thermal stability of Arc repressor.

The thermal stability of the Arc repressor dimer normally increases with concentration because protein folding and subunit association are thermodynamically coupled. At Arc concentrations above 100 microM, however, thermal denaturation remains reversible and cooperative but tm does not continue to increase. In this concentration regime, thermally denatured Arc shows significantly reduced secondary structure and no evidence of a tightly packed core, but light scattering and fluorescence polarization studies indicate that the protein is dimeric. Higher order denatured oligomers are not observed and the stability of the non-native dimer is reduced by Arc mutations, indicating that non-native dimerization involves specific interactions between Arc subunits.

Thermodynamic investigation of the association of ethidium, propidium and bis-ethidium to DNA hairpins.

We have used a combination of calorimetric and spectroscopic techniques to investigate the association of the bis-intercalator ethidium homodimer (bis-ethidium) to short DNA hairpins with sequences: d(GCGCT5GCGC) and d(CGCGT5CGCG). The helix-coil transition of each hairpin, investigated by UV and calorimetric melting protocol, takes place in monomolecular two-state transitions with characteristic enthalpies of approximately 37 kcal mol-1 for disrupting the four dG-dC base pairs of the hairpin stems. Deconvolution of the bis-ethidium-hairpin calorimetric titration curves indicate that each hairpin contains two distinct binding sites for the ligand: a high affinity site in the stem (Kb approximately 10(7)) that accommodates one bis-ethidium molecule and a lower affinity site (Kb approximately 10(6)) located probably at the loop that accommodates two bis-ethidium molecules. The overall stoichiometries of three ligands per hairpin are in agreement with those obtained in continuous variation experiments using visible spectroscopy. The interaction of bis-ethidium for each type of sites results in enthalpy driven reactions, with average binding enthalpies, delta Hb, of -13.1 and -12.1 kcal mol-1 for the stem and loop sites, respectively. Comparison to the thermodynamic profiles of ethidium and propidium binding reveals that the bis-ethidium binding to the stem site of each hairpin has a more favorable free energy term of -1.4 kcal mol-1 and more favorable enthalpy of -4.2 kcal mol-1. These suggest that only one phenanthridine ring of bis-ethidium intercalates in the stem, while the second planar ring is exposed to solvent or weakly associated to the surface of DNA.

Interaction of minor groove ligands to an AAATT/AATTT site: correlation of thermodynamic characterization and solution structure.

A combination of circular dichroism spectroscopy, titration calorimetry, and optical melting has been used to investigate the association of the minor groove ligands netropsin and distamycin to the central A3T2 binding site of the DNA duplex d(CGCAAATTGGC).d(GCCAATTTGCG). For the complex with netropsin at 20 degrees C, a ligand/duplex stoichiometry of 1:1 was obtained with Kb approximately 4.3 x 10(7) M-1, delta Hb approximately -7.5 kcal mol-1, delta Sb approximately 9.3 cal K-1 mol-1, and delta Cp approximately 0. Previous NMR studies characterized the distamycin complex with A3T2 at saturation as a dimeric side-by-side complex. Consistent with this result, we found a ligand/duplex stoichiometry of 2:1. In the current study, the relative thermodynamic contributions of the two distamycin ligands in the formation of this side-by-side complex (2:1 Dst.A3T2) were evaluated and compared with the thermodynamic characteristics of netropsin binding. The association of the first distamycin molecule of the 2:1 Dst.A3T2 complex yielded the following thermodynamic profile: Kb approximately 3.1 x 10(7) M-1, delta Hb = -12.3 kcal mol-1, delta Sb = -8 cal K-1 mol-1, and delta Cp = -42 cal K-1 mol-1. The binding of the second distamycin molecule occurs with a lower Kb of approximately 3.3 x 10(6) M-1, a more favorable delta Hb of -18.8 kcal mol-1, a more unfavorable delta Sb of -34 cal K-1 mol-1, and a higher delta Cp of -196 cal K-1 mol-1. The latter term indicates an ordering of electrostricted and structural water molecules by the complexes. These results correlate well with the NMR titrations and are discussed in context of the solution structure of the 2:1 Dst.A3T2 complex.

Differential hydration of dA.dT base pairs in parallel-stranded DNA relative to antiparallel DNA.

Parallel-stranded DNA is a novel double-stranded helical form of DNA. Its secondary structure is established by reverse Watson-Crick base pairing between the bases of the complementary strands forming a double helix with equivalent grooves. We have used a combination of magnetic suspension densitometry and isothermal titration calorimetry to obtain complete thermodynamic profiles for the formation of two DNA 25 mer duplexes. The duplexes contain exclusively dA.dT base pairs in either parallel (ps-d1.D2) or antiparallel (aps-D1.D3) orientation. At 15 degrees C, the formation of each duplex is accompanied by favorable free-energy terms resulting from the partial compensation of favorable exothermic enthalpies and unfavorable entropies and by an uptake of both counterions and water molecules. By taking into account the contribution of single-strand base-stacking interactions and using the formation of the aps-D1.D3 duplex as a reference state to establish a thermodynamic cycle in which the similar single strands cancel out, we obtained a delta delta G zero term of +10 kcal mol-1 duplex formed that results from a partial differential enthalpy-entropy compensation of +32 kcal mol-1 and a delta delta V of 257 mL mol-1. The positive sign of this enthalpy-entropy compensation together with the marginal differential counterion uptake of 0.2 mol of Na+/mol of duplex is characteristic of processes driven by differential hydration and strongly suggests that the parallel duplex is much less hydrated than its antiparallel counterpart by approximately 4 mol of water/mol of base pair.

Thermodynamics of DNA hairpins: contribution of loop size to hairpin stability and ethidium binding.

A combination of calorimetric and spectroscopic techniques was used to evaluate the thermodynamic behavior of a set of DNA hairpins with the sequence d(GCGCTnGCGC), where n = 3, 5 and 7, and the interaction of each hairpin with ethidium. All three hairpins melt in two-state monomolecular transitions, with tm's ranging from 79.1 degrees C (T3) to 57.5 degrees C (T7), and transition enthalpies of approximately 38.5 kcal mol-1. Standard thermodynamic profiles at 20 degrees C reveal that the lower stability of the T5 and T7 hairpins corresponds to a delta G degree term of +0.5 kcal mol-1 per thymine residue, due to the entropic ordering of the thymine loops and uptake of counterions. Deconvolution of the ethidium-hairpin calorimetric titration curves indicate two sets of binding sites that correspond to one ligand in the stem with binding affinity, Kb, of approximately 1.8 x 10(6) M-1, and two ligands in the loops with Kb of approximately 4.3 x 10(4) M-1. However, the binding enthalpy, delta Hb, ranges from -8.6 (T3) to -11.6 kcal mol-1 (T7) for the stem site, and -6.6 (T3) to -12.7 kcal mol-1 (T7) for the loop site. Relative to the T3 hairpin, we obtained an overall thermodynamic contribution (per dT residue) of delta delta Hb = delta(T delta Sb) = -0.7(5) kcal mol-1 for the stem sites and delta delta Hb = delta(T delta Sb) = -1.5 kcal mol-1 for the loop sites. Therefore, the induced structural perturbations of ethidium binding results in a differential compensation of favorable stacking interactions with the unfavorable ordering of the ligands.

Contribution of loops and nicks to the formation of DNA dumbbells: melting behavior and ligand binding.

We have evaluated the thermodynamic contribution of thymine loops and nicks to the overall stability of double-helical DNA by investigating (1) the melting behavior of two unligated DNA dumbbells and their corresponding core duplexes and (2) the association of netropsin to the central core of four A.T base pairs of these molecules. Temperature-dependent UV absorption and differential scanning calorimetry techniques have been used to characterize the helix-coil transitions of all four deoxyoligonucleotide duplexes. In 10 mM NaP(i) buffer at pH 7.0, all transitions were monophasic. The dumbbells melt with transition temperatures, Tm, independent of strand concentration, while each duplex melts with transition temperature dependence on strand concentration, characteristic of mono- and bimolecular processes, respectively. The Tm's for the dumbbells correspond to those of single hairpins containing only four base pairs in the stem. We obtain dTm/d log [Na+] values of 10.9-12.5 degrees C for these molecules, which correspond to similar counterion releases and suggest helical structures with similar charge densities and helical strandedness. Standard thermodynamics profiles at 5 degrees C reveal that the favorable free energy of forming these ordered structures results from the partial compensation of favorable enthalpies with unfavorable entropies. The stabilization of the dumbbells relative to the core duplexes is enthalpic, due to extra stacking of the nearest loop thymines on the G.C base pairs at both ends of the stem.(ABSTRACT TRUNCATED AT 250 WORDS)

Volume changes correlate with entropies and enthalpies in the formation of nucleic acid homoduplexes: differential hydration of A and B conformations.

We have used a combination of densimetric, calorimetric, and uv absorption techniques to obtain a complete thermodynamic characterization for the formation of nucleic acid homoduplexes of known sequence and conformation. The volume change delta V accompanying the formation of four duplexes was interpreted to reflect changes in hydration based on the electrostriction phenomenon. In 10 mM sodium phosphate buffer at pH 7, the magnitude of the measured delta V's ranged from -2.0 to +7.2 ml/mol base pair and followed the order of poly(rA).poly(dT) approximately poly(dA).poly(dT) < poly(rA).poly(dU) approximately poly(rA).poly(rU). Inclusion of 100 mM NaCl in the same buffer gave the range of -17.4 to -2.3 mL/mol base pair and the following order: poly(dA).poly(dT) < poly(rA).poly(dT) < poly(rA).poly(rU) approximately poly(rA).polyr(dU). Standard thermodynamic profiles of forming these duplexes from their corresponding complementary single strands indicated similar free energies that resulted from the compensation of favorable enthalpies with unfavorable entropies along with a similar counterion uptake at both ionic strengths. The differences in these compensating effects of entropy and enthalpy correlated very well with the volume change measurements in a manner suggesting that the homoduplexes in the B conformation are more hydrated than are those in the A conformation. Moreover, the increased thermal stability of these homoduplexes resulted from an increase in the salt concentration corresponding to larger hydration levels as reflected by the delta V results.

Differential hydration of homopurine sequences relative to alternating purine/pyrimidine sequences.

The minor groove ligand distamycin A has been used to probe the relative hydration of the minor groove of eight synthetic polynucleotides of known sequence and composition. A combination of densimetric, calorimetric, and temperature-dependent spectroscopic techniques have been used to obtain complete thermodynamic profiles (delta Gzero, delta Hzero, delta Szero, and delta Vzero) for the association of distamycin A to all polymer duplexes. In 10 mM phosphate buffer, pH 7, binding of the drug to each of the polymeric duplexes resulted in characteristic negative changes in both the volume and enthalpy. Although the binding constants were found to be identical for pairs of isomer polynucleotides having identical compositions but different sequences, the values of delta Hzero, delta Szero, and delta Vzero of each such pair were remarkably different. The entropy changes were found to roughly parallel the volume changes; no such trend was seen between delta Hzero and delta Vzero. The data support the hypothesis that the volume changes observed for these systems reflect the coulombic-hydration contribution to the entropy. The heteropolymer duplexes generated much larger exothermic contributions, less favorable entropies and larger volume contractions than did the corresponding homopolymer duplexes of identical composition, and strongly suggest that polynucleotides with homopurine sequences are more hydrated than polynucleotides with alternating purine/pyrimidine sequences. In addition, it was found that duplexes containing guanine sharply reduced the affinity for the drug, also lowering the exothermicity but raising the entropy. This may be explained by the presence of an amino group in the minor groove that prevents hydrogen bonding. Substitution of the guanine with inosine reversed this trend in the thermodynamic properties. Furthermore, substitution of poly(dA) for poly(rA) in a duplex produced a similar reduction in the affinity, while raising the exothermic contribution and greatly reducing the favorable entropy effect in agreement with an apparent increase in the hydration state.

Coupling of sequential transitions in a DNA double hairpin: energetics, ion binding, and hydration.

In an effort to evaluate the relative contributions of sequence, ion binding, and hydration to the thermodynamic stability of nucleic acids, we have investigated the melting behavior of a double hairpin and that of its component single hairpins. Temperature-dependent UV absorption and differential scanning calorimetry techniques have been used to characterize the helix-coil transitions of three deoxyoligonucleotides: d(GTACT5GTAC), d(GCGCT5GCGC), and d(GCGCT5GCGCGTACT5GTAC). The first two oligomers melt with transition temperatures equal to 28 and 69 degrees C, respectively, in 10 mM dibasic sodium phosphate at pH 7.0. The Tm's are independent of strand concentration, strongly indicating the presence of single-stranded hairpin structures at low temperatures. The third oligomer, with a sequence corresponding to the joined sequences of the first two oligomers, melts with two apparently independent monomolecular transitions with Tm's of 41 and 69 degrees C. These transitions correspond to the melting of a double hairpin. In the salt range of 10-100 mM in NaCl, we obtain average enthalpies of 24 and 38 kcal/mol for the transitions in the single-hairpin molecules. Each transition in the double hairpin has an enthalpy of 32 kcal/mol. In addition, dtm/d log [Na+] for the transitions are 4.1 and 4.7 degrees C for the single hairpins and 12.6 and 11.2 degrees C for each transition in the double hairpin. The differential ion binding parameter between the double hairpin and that of the sum of single hairpins is roughly equal to 1.1 mol of Na+ ions/mol of double hairpin and is consistent with an increase in the electrostatic behavior of the stem phosphates of this molecule.