We report a direct determination of the thermodynamic contribution that DNA single-stranded order makes to DNA duplex formation. By using differential scanning calorimetry (DSC) and temperature-dependent UV absorbance spectroscopy, we have characterized thermodynamically the thermally induced disruption of the 13-mer duplex [d(CGCATGAGTACGC) of -56.4 kcal/mol for duplex formation at 25°C using isothermal batchmixing calorimetry. This duplex formation enthalpy of -56.4 kcal/mol at 250C is very different in magnitude from the duplex disruption enthalpy of 117.0 kcal/mol measured at 74VC by DSC. Since the DSC measurement reveals the net transition heat capacity change to be close to zero, we interpret this large disparity between the enthalpies ofduplex disruption and duplex formation as reflecting differences in the single-stranded structures at 250C (the initial states in the isothermal mixing experiment) and the single-stranded structures at W80C (the final states in the DSC experiment). In fact, the enthalpy for duplex formation at 250C (-56.4 This feature of single-stranded structure near room temperature can reduce significantly the enthalpic driving force one might predict for duplex formation from nearest-neighbor data, since such data generally are derived from measurements in which the single strands are in their random-coil states. Consequently, potential contributions from single-stranded structure must be recognized and accounted for when designing hybridization experiments and when using isothermal titration and/or batch mixing techniques to study the formation of duplexes and higher-order DNA structures (e.g., triplexes, tetraplexes, etc.) from their component single strands.The existence of ordered structure in oligo-and polynucleotide single strands has been known for some time and has stimulated considerable research designed to elucidate the detailed nature of the forces that give rise to such singlestranded order, as well as the influence of this order on duplex formation (refs. 1-8 and references cited in ref. 8).Most early studies made use of optical techniques such as ultraviolet (UV) and infrared spectroscopy, circular dichroism (CD), and optical rotary dispersion. Other experimental approaches such as NMR, intrinsic viscosity, and sedimentation coefficients also were used, albeit to a lesser extent. In most, but not all, of these studies, the authors interpret their data to be consistent with the existence of some ordered single-stranded structure. Temperature-dependent studies of various equilibrium properties showed the single-stranded structures to "melt" over rather broad temperature ranges compared with higher-order nucleic acid structures (e.g., duplexes, triplexes, etc.). Thermodynamic data were derived indirectly from the temperature-dependent properties by assuming a model for these melting processes (4, 9-13). However, because of the approximations associated with such van't Hoffanalyses, as well as difficulties in defining the extent of order in the initial and ...

Recent theoretical studies performed on the folding/unfolding mechanism of the model telomeric human DNA, 5'-AGGGTTAGGGTTAGGGTTAGGG-3' (Tel22), have indicated that in the presence of K(+) ions Tel22 folds into two hybrid G-quadruplex structures characterized by one double and two reversal TTA loops arranged in a different way. They predicted a new unfolding pathway from the initial mixture of hybrid G-quadruplexes via the corresponding intermediate triplex structures into the final, fully unfolded state. Significantly, no experimental evidence supporting the suggested pathway has been reported. In the current work, we performed a comprehensive global thermodynamic analysis of calorimetric (DSC, ITC) and spectroscopic (CD) data obtained on monitoring the folding/unfolding of Tel22 induced by changes of temperature and K(+) concentration. We show that unfolding of Tel22 may be described as a monomolecular equilibrium three-state process that involves thermodynamically distinguishable folded (F), intermediate (I), and unfolded (U) state. Considering that calorimetric methods cannot distinguish between energetically similar G-quadruplex or triplex conformations predicted by the theoretical model one can conclude that our results represent the first experimental support of the suggested unfolding/folding mechanism of Tel22. This conclusion is confirmed by the fact that the estimated number of K(+) ions released upon each unfolding step in our thermodynamic model agrees well with the corresponding values predicted by the theoretical model and that the observed changes in enthalpy, entropy, and heat capacity accompanying the F → I and I → U transitions can be reasonably explained only if the intermediate state I is considered to be a triplex structural conformation.

We report a complete thermodynamic characterization of the impact of abasic and anucleosidic lesions on the stability, conformation, and melting behavior of a DNA duplex. The requisite thermodynamic data were obtained by using a combination of spectroscopic and calorimetric techniques to investigate helix-to-coil transitions in a family of DNA duplexes of the form d(CGCATGAGTACGC)-d(GCGTA-CXCATGCG), where X corresponds to a thymidine residue in the parent Watson-Crick duplex and to an abasic or anucleosidic site in the modified duplexes. The data derived from these studies reveal that incorporation of an abasic site into a DNA duplex dramatically reduces the duplex stability, transition enthalpy, and transition entropy. The magnitudes of these lesion-induced effects are greater than one would expect based on simple nearest-neighbor considerations. Nearly identical thermodynamic data are obtained when the modified duplex contains an anucleosidic site rather than an abasic site. This observation suggests that the thermodynamic impact of these lesions primarily results from removal of the base rather than the sugar ring. Significantly, the melting cooperativities of the abasic and anucleosidic derivatives are identical with each other and with the corresponding unmodified Watson-Crick parent duplex. This result suggests that the phosphodiester backbone, rather than the base-sugar network, serves as the primary propagation path for the communication of cooperative melting effects. We propose molecular interpretations for the thermodynamic data based on the structural picture that has emerged from the NMR studies of Patel and coworkers on the same family of modified and unmodified DNA duplexes

Thermal denaturation of equinatoxin II (EqTxII) in glycine buffer solutions (pH 1.1, 2.0, 3.0, and 3.5) and in triple distilled water (pH 5.5-6.0) was examined by differential scanning calorimetry, UV and CD spectroscopy and fluorescence emission spectroscopy of the added hydrophobic fluorescent probe ANS. At pH 5.5-6.0 and at temperatures below 60 degrees C, the protein exists in a native state characterized by a pronounced tertiary structure, a beta-rich secondary structure and a low degree of ANS-binding. At higher temperatures, it undergoes a two-state conformational transition, (delta H degree)VH = (delta H degree)DSC, into an unfolded state, which is characterized by a complete collapse of its tertiary structure and an incomplete denaturation of its secondary structure. At acidic pH, the EqTxII temperature-induced conformational transition appears at lower temperatures as non-two-state transition accompanied by the formation of an intermediate state which shows characteristics of molten globules, i.e., absence of defined tertiary structure, increase in alpha-rich secondary structure, and high affinity for ANS. At pH 2.0, the low-temperature initial state of EqTxII is already partially denatured; the tertiary structure is partially disrupted, and a pronounced inequality (delta H degree)VH > (delta H degree)DSC is observed. At pH value of 1.1 and below 60 degrees C, EqTxII exists in a stable acid-denatured compact state which shows all the characteristics of a molten globule, which even at 95 degrees C is not completely denatured. According to numerous studies on the pore forming toxins, such acid-denatured compact states may contribute to the protein's ability to penetrate into biological membranes.

The degree and the enthalpy of binding of dodecyl-and cetylpyridlnium (DP+ and CP+) cations to poly(styrenesulfonate) anion at 25 °C in aqueous solutions containing excess of NaCl are reported. The degree of binding has been determined by using a potentiometric technique based on surfactant-cation-selective membrane electrodes. The appreciable binding of both surfactants starts at the total detergent concentration about 10"5 mol/L which is a few orders of magnitude below the critical micelle concentrations, cmc, and the binding of CP+ cation is almost complete. Above this concentration the enthalpy of binding, calculated per mole of bound surfactant cations, is practically constant and for the DP+ cation (for which the corresponding literature calorimetric data exist) approximately equal to the enthalpy of micellization of dodecylpyridinium iodide. From the solution of the Poisson-Boltzmann equation for the cell model of a polyelectrolyte solution with two kinds of monovalent counterions of different size, the distribution of counterions around the polyion is calculated. The calculations show that the local concentration of the surfactant counterions at the surface of the polyion exceeds the average concentration by a factor of about 1800, at the experimental conditions (CP = 5 X 10"4 monomol/L). On the basis of the experimental results and these calculations it is concluded that many small surfactant aggregates are formed in the polyion domain, much below the cmc. This finding agrees with the results of previous photochemical studies of polyelectrolyte-surfactant interactions.

Titration calorimetry was employed to study the thermodynamic parameters of micellization of N-undecyl-, N-tridecyl-, N-tetradecyl-, and N-pentadecylpyridinium bromides and tetradecyl-and hexadecyltrimethylammonium bromides in salt-free aqueous solutions at 25°C. For this purpose a model equation for the calorimetric titration curve based on the mass action model of the micellization process was derived. As adjustable parameters it contains the aggregation number n, the degree of micelle ionization p/n, and the enthalpy of micellization ∆H M . Critical micelle concentrations cmc were determined by applying the Philips criterion to the experimental titration curves and so obtained cmc values are in excellent agreement with the literature data. Fitting of the model titration curves to those obtained experimentally shows that this procedure gives reliable values only for ∆H M while it fails to provide any useful information on the p/n data. In addition, for surfactants with short alkyl chains and thus with low aggregation numbers (n < ≈50) this fitting method provides also aggregation numbers that are close to those reported in the literature.

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Isothermal calorimetric titrations (ITC), circular dichroism (CD), and UV spectroscopy have been employed to investigate and quantify binding of the enantiomers of N-3,5-dinitrobenzoyl-leucine (DNB-Leu) and nonchiral N-3,5-dinitrobenzoyl-glycine (DNB-Gly), denoted as selectands (SAs) to the following chiral selectors (SOs):  quinine (QN), quinidine (QD) and their derivatives O9-tert-butylcarbamoyl quinine (t-BuCQN) and O9-tert-butylcarbamoyl quinidine (t-BuCQD). The results reveal that DNB-Leu binds to all SOs in a 1:1 association mode. Although DNB-Leu exhibits higher affinity for QN and QD than for t-BuCQN and t-BuCQD, no preferential binding of any of the two DNB-Leu enantiomers to QN or QD was observed. By contrast, t-BuCQN binds (S)-DNB-Leu with high enantioselectivity (K b,S/K b,R ≈ 10), whereas the t-BuCQD derivative shows similarly high selectivity for the (R)-DNB-Leu enantiomer (K b,R/K b,S ≈ 10). The results of optical (CD, UV) titrations of t-BuCQN with (S)-DNB-Leu and t-BuCQD with (R)-DNB-Leu are fully consistent with those obtained from the corresponding calorimetric titrations. The induced CD spectra of (S)-DNB-Leu-t-BuCQN and (R)-DNB-Leu-t-BuCQD ionic complexes display bands of opposite sign indicating that binding of DNB-Leu enantiomers within the SOs molecules occurs at well-defined domains in a “pseudo-enantiomeric fashion” (Lämmerhofer and Lindner, J. Chromatog. 1996, 741, 33). The relative binding constants derived from ITC, UV, and CD titrations are in good agreement with the enantioseparation factors observed with the corresponding immobilized SO versions under HPLC conditions in prior studies. The thermodynamic analysis shows that the ion-pair formation between cinchona alkaloid type SOs and DNB-leucine is a strongly enthalpy-driven process ( up to −38 kJ/mol), accompanied by unfavorable entropic contributions ( up to −15 kJ/mol). The observed highly exothermic values result most likely from the attractive noncovalent intermolecular interactions, such as van der Waals interactions, hydrogen bonding and π−π interactions, whereas the negative entropy contributions apparently reflect the generation of highly ordered bimolecular ionic associates.