Complete Thermodynamic Profiles Research Articles

Thermodynamic Insights into Phosphonate Binding in Metal-Azolate Frameworks.

Organophosphorus chemicals, including chemical warfare agents (CWAs) and insecticides, are acutely toxic materials that warrant capture and degradation. Metal-organic frameworks (MOFs) have emerged as a class of tunable, porous, crystalline materials capable of hydrolytically cleaving, and thus detoxifying, several organophosphorus nerve agents and their simulants. One such MOF is M-MFU-4l (M = metal), a bioinspired azolate framework whose metal node is composed of a variety of divalent first-row transition metals. While Cu-MFU-4l and Zn-MFU-4l are shown to rapidly degrade CWA simulants, Ni-MFU-4l and Co-MFU-4l display drastically lower activities. The lack of reactivity was hypothesized to arise from the strong binding of the phosphate product to the node, which deactivates the catalyst by preventing turnover. No such study has provided detailed insight into this mechanism. Here, we leverage isothermal titration calorimetry (ITC) to monitor the binding of an organophosphorus compound with the M-MFU-4l series to construct a complete thermodynamic profile (Ka, ΔH, ΔS, ΔG) of this interaction. This study further establishes ITC as a viable technique to probe small differences in thermodynamics that result in stark differences in material properties, which may allow for better design of first-row transition metal MOF catalysts for organophosphorus hydrolysis.

Roles of electrostricted and structural hydration in the stability of nucleic acid duplexes.

To understand the mechanisms that govern the many biological roles of nucleic acids, it is essential to have a complete physical description of the folding of nucleic acids, including ion and water binding. To investigate the role of water in the stability of nucleic acid duplexes, we use biophysical techniques such as differential scanning calorimetry (DSC and PPC), density, ultrasound and temperature-dependence UV spectroscopy to measure complete thermodynamic profiles for the unfolding of DNA hairpins and duplexes containing chemical modifications. The favorable folding of a DNA duplex (negative ΔGo) results from a compensation between a favorable ΔH and unfavorable TΔS contributions. The resulting compensating temperatures are characteristic of the type of hydration, electrostricted or structural. DSC and UV melting curves of DNA duplexes as a function of salt and osmolyte concentrations show ion and water releases. Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of both counterions and water molecules. Furthermore, the comparison of the signs of ΔΔGo (or ΔΔH-Δ(TΔS) compensation) with ΔΔV of the Hess cycles for pairs of duplexes with and without a chemical modification (base pair, bulge, mismatch or adduct), allow us to indicate the type of hydration of the chemical modification: similar signs indicate electrostricted hydration while opposite signs indicate structural hydration. The main conclusion is the incorporation of stabilizing chemical modifications in DNA is accompanied by an immobilization of electrostricted water while the incorporation of destabilizing modifications immobilizes structural water. Supported by Grant MCB-1912587 from National Science Foundation.

Single-molecule calorimeter and free energy landscape

The precise measurement of thermodynamic and kinetic properties for biomolecules provides the detailed information for a multitude of applications in biochemistry, biosensing, and health care. However, sensitivity in characterizing the thermodynamic binding affinity down to a single molecule, such as the Gibbs free energy ([Formula: see text]), enthalpy ([Formula: see text]), and entropy ([Formula: see text]), has not materialized. Here, we develop a nanoparticle-based technique to probe the energetic contributions of single-molecule binding events, which introduces a focused laser of optical tweezer to an optical path of plasmonic imaging to accumulate and monitor the transient local heating. This single-molecule calorimeter uncovers the complex nature of molecular interactions and binding characterizations, which can be employed to identify the thermodynamic equilibrium state and determine the energetic components and complete thermodynamic profile of the free energy landscape. This sensing platform promises a breakthrough in measuring thermal effect at the single-molecule level and provides a thorough description of biomolecular specific interactions.

Unveiling the binding interaction of zinc (II) complexes of homologous Schiff‐base ligands on the surface of BSA protein: A combined experimental and theoretical approach

Four new zinc (II) complexes [Zn (HL1H)Br2] (1), [Zn (HL1H)Cl2] (2), [Zn2(HL2)Br3] (3), and [Zn (HL2)Cl] (4) have been synthesized by adopting template synthetic strategy and utilizing two homologous Schiff base ligands (H2L1 = 4‐bromo‐2‐{[2‐(2‐hydroxyethylamino)‐ethylimino]‐methyl}‐6‐methoxyphenol, H2L2 = 4‐bromo‐2‐{[3‐(2‐hydroxyethylamino)propylimino]methyl}‐6‐methoxyphenol), differing in one ‐CH2‐ unit in the ligating backbone, by adopting template synthetic strategy. All the complexes have been characterized by single crystal X‐ray diffraction analysis as well as by other routine physicochemical techniques. Ligand mediated structural variations have been observed and rationalized by density functional theoretical (DFT) calculations. Interaction of the complexes 1–4 with Bovine Serum Albumin protein (BSA) has been studied by different spectroscopic techniques. A complete thermodynamic profile (ΔHo, ΔSo and ΔGo) was evaluated initially from the change in absorption and fluorescence spectra upon addition of BSA to the complexes. Appreciable binding constant values in the range ~ 0.94–4.51 × 104 M−1 indicate efficient binding tendency of the complexes to BSA with the sequence 1 ≅ 2 > 3 ≅ 4. Circular dichroism (CD), isothermal calorimetric titration experiments, molecular docking and molecular dynamics have been performed to gain deep insight into the binding regions of complex 1 to BSA. Experimental evidences suggest an interaction of zinc complexes at the surface of BSA protein and this particular binding has been exploited to determine unknown concentration of BSA protein. For this purpose complex 1 was explored as a BSA protein quantification tool.

Analysis of the HIV-1 Genomic RNA Dimerization Initiation Site Binding to Aminoglycoside Antibiotics Using Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) provides a sensitive, powerful, and accurate tool to suitably analyze the thermodynamic of RNA binding events. This approach does not require any modification or labeling of the system under analysis and is performed in solution. ITC is a very convenient technique that provides an accurate determination of binding parameters, as well as a complete thermodynamic profile of the molecular interactions. Here we show how this approach can be used to characterize the interactions between the dimerization initiation site (DIS) RNA localized within the HIV-1 viral genome and aminoglycoside antibiotics. Our ITC study showed that the 4,5-disubstituted 2-desoxystreptamine (2-DOS) aminoglycosides can bind the DIS with a nanomolar affinity and a high specificity.

Tinkering with Binding Polynomials in Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) has become the preferred experimental technique for characterizing intermolecular interactions between biological molecules. Among the several advantages, the use of natural non-labeled molecules and the determination of the complete thermodynamic profile for the interaction in solution remain as the primary features that have promoted ITC to the forefront of experimental biophysics. The experimental design in ITC may range from studying a simple direct binary macromolecule-ligand interaction to studying the homotropic or heterotropic cooperative effect between ligands when interacting with a given macromolecule. The theory of the binding polynomial has proven to be an appropriate unifying framework for handling the complexities that can be encountered when studying macromolecule-ligand interactions, though it has been deemed troublesome. The goal of this chapter is to provide a quite simple and widely available set of training experiments aimed at mastering the formalism of the binding polynomial applied to isothermal titration calorimetry.

Berenil Binds Tightly to Parallel and Mixed Parallel/Antiparallel G-Quadruplex Motifs with Varied Thermodynamic Signatures.

Diminazene, DMZ, (or berenil) has been reported as a tight binder of G-quadruplexes. G-Quadruplex structures are often located in the promotor regions of oncogenes and may play a regulatory role in gene expression based on the stability of the folding topology. In this study, attempts have been made to characterize the specificity of DMZ binding toward multiple G-quadruplex topologies or foldamers. Mutant sequences of the G-quadruplex forming promotor regions of several oncogenes were designed to exhibit restricted loop lengths and folding topologies. Circular dichroism was used to confirm the quadruplex topology of mutant BCL2, KRAS, and c-MYC sequences, human telomere (Na+ and K+) G-quadruplexes and their complexes with DMZ and analogs thereof. Isothermal titration calorimetry was used to generate a complete thermodynamic profile (ΔG, ΔH, −TΔS) for the formation of DMZ and analog complexes with the target G-quadruplexes. DMZ binds to parallel and/or mixed parallel/antiparallel quadruplex DNA motifs with stoichiometries up to 8:1 and via three binding modes with varying affinities. In the case of the parallel G-quadruplexes, with the exception of the long-looped c-MYC mutant, the highest affinity binding event (mode 1) is driven by enthalpy. DMZ binding to the long-looped c-MYC mutant exhibits a very favorable entropy change in addition to a moderately favorable enthalpy change. Mode 1 binding to the antiparallel and mixed parallel/antiparallel hTel quadruplexes is also driven by favorable enthalpy changes. In all cases, the intermediate DMZ affinity binding (mode 2) is driven almost entirely by entropy, with small or unfavorable enthalpic contributions. The weakest binding event (mode 3) is also entropically driven with small or moderate enthalpic contributions.

Energetics, Ion, and Water Binding of the Unfolding of AA/UU Base Pair Stacks and UAU/UAU Base Triplet Stacks in RNA.

Triplex formation occurs via interaction of a third strand with the major groove of double-stranded nucleic acid, through Hoogsteen hydrogen bonding. In this work, we use a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry to determine complete thermodynamic profiles for the unfolding of polyadenylic acid (poly(rA))·polyuridylic acid (poly(rU)) (duplex) and poly(rA)·2poly(rU) (triplex). Our thermodynamic results are in good agreement with the much earlier work of Krakauer and Sturtevant using only UV melting techniques. The folding of these two helices yielded an uptake of ions, Δ nNa+ = 0.15 mol Na+/mol base pair (duplex) and 0.30 mol Na+/mole base triplet (triplex), which are consistent with their polymer behavior and the higher charge density parameter of triple helices. The osmotic stress technique yielded a release of structural water, Δ nW = 2 mol H2O/mol base pair (duplex unfolding into single strands) and an uptake of structural water, Δ nW = 2 mol H2O/mole base pair (triplex unfolding into duplex and a single strand). However, an overall release of electrostricted waters is obtained for the unfolding of both complexes from pressure perturbation calorimetric experiments. In total, the Δ V values obtained for the unfolding of triplex into duplex and a single strand correspond to an immobilization of two structural waters and a release of three electrostricted waters. The Δ V values obtained for the unfolding of duplex into two single strands correspond to the release of two structural waters and the immobilization of four electrostricted water molecules.

The Irving-Williams series and the 2-His-1-carboxylate facial triad: a thermodynamic study of Mn2+, Fe2+, and Co2+ binding to taurine/α-ketoglutarate dioxygenase (TauD).

Taurine/α-ketoglutarate (αKG) dioxygenase (TauD) is an E. coli nonheme Fe2+- and αKG-dependent metalloenzyme that catalyzes the hydroxylation of taurine, leading to the production of sulfite. The metal-dependent active site in TauD is formed by two histidine and one aspartate that coordinating to one face of an octahedral coordination geometry, known as the 2-His-1-carboxylate facial triad. This motif is found in many nonheme Fe2+ proteins, but there is limited information on the thermodynamic parameters that govern metal-ion binding to this site. Here, we report data from calorimetry and related biophysical techniques to generate complete thermodynamic profiles of Mn2+ and Co2+ binding to TauD, and these values are compared to the Fe2+ data reported earlier Henderson et al. (Inorg Chem 54: 2278-2283, 2015). The buffer-independent binding constants (K) were measured to be 1.6 × 106, 2.4 × 107, and 1.7 × 109, for Mn2+, Fe2+, and Co2+, respectively. The corresponding ΔG° values were calculated to be -8.4, -10.1, and -12.5kcal/mol, respectively. The metal-binding enthalpy changes (ΔH) for these binding events are -11.1 (± 0.1), -12.2 (± 0.1), and -16.0 (± 0.6) kcal/mol, respectively. These data are fully consistent with the Irving-Williams series, which show an increasing affinity for transition metal ions across the periodic table. It appears that the periodic increase in affinity, however, is a result of a complicated summation of enthalpy terms (including favorable metal-ion coordination processes and unfavorable ionization events) and related entropy terms.

Differential Scanning Calorimetry - A Method for Assessing the Thermal Stability and Conformation of Protein Antigen.

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural “fingerprint” that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; ∆H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential Scanning Calorimetry — A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural “fingerprint” that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; ∆H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Intrinsic local destabilization of the C-terminus predisposes integrin α1 I domain to a conformational switch induced by collagen binding.

Integrin-collagen interactions play a critical role in a myriad of cellular functions that include immune response, and cell development and differentiation, yet their mechanism of binding is poorly understood. There is increasing evidence that conformational flexibility assumes a central role in the molecular mechanisms of protein-protein interactions and here we employ NMR hydrogen-deuterium exchange (HDX) experiments to explore the impact of slower timescale dynamic events. To gain insight into the mechanisms underlying collagen-induced conformational switches, we have undertaken a comparative study between the wild type integrin α1 I and a gain-of-function E317A mutant. NMR HDX results suggest a relationship between regions exhibiting a reduced local stability in the unbound I domain and those that undergo significant conformational changes upon binding. Specifically, the αC and α7 helices within the C-terminus are at the center of such major perturbations and present reduced local stabilities in the unbound state relative to other structural elements. Complementary isothermal titration calorimetry experiments have been performed to derive complete thermodynamic binding profiles for association of the collagen-like triple-helical peptide with wild type α1 I and E317A mutant. The differential energetics observed for E317A are consistent with the HDX experiments and support a model in which intrinsically destabilized regions predispose conformational rearrangement in the integrin I domain. This study highlights the importance of exploring different timescales to delineate allosteric and binding events.

Engineering and characterization of fluorogenic glycine riboswitches

A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch library. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570 μM. Furthermore, the kinetic glycine binding (kon), and dissociation (koff) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. kon and koff were in the order of 10−3s−1 and 10−2s−1, respectively. Conclusively, we demonstrate that systematic screening of synthetic and natural linked RNA parts with microfluidic chip technology is an effective approach to rapidly generate fluorogenic metabolite riboswitches with a broad range of biophysical response properties.

Application of isothermal titration calorimetry as a tool to study natural product interactions.

Covering: up to February 2015Over the past twenty-five years, isothermal titration calorimetry (ITC) has become a potent tool for the study a great variety of molecular interactions. This technique is able to provide a complete thermodynamic profile of an interaction process in a single experiment, with a series of advantages in comparison to other comparable techniques, such as less amount of sample or no need of chemical modification or labelling. It is thus not surprising that ITC has been applied to study the manifold types of interactions of natural products to get new insights into the molecular key factors implied in the complexation process of this type of compounds. This review provides an overview over the applications of ITC as a potent tool to investigate interactions of natural products with proteins, nucleic acids, oligosaccharides, and other types of receptors. The examples have been selected depending on the impact that this technique had during the investigation and revision of the interactions involved in the bioactivity of a compound, lead optimization or technical applications.

A microfabrication-based approach to quantitative isothermal titration calorimetry

Isothermal titration calorimetry (ITC) directly measures heat evolved in a chemical reaction to determine equilibrium binding properties of biomolecular systems. Conventional ITC instruments are expensive, use complicated design and construction, and require long analysis times. Microfabricated calorimetric devices are promising, although they have yet to allow accurate, quantitative ITC measurements of biochemical reactions. This paper presents a microfabrication-based approach to integrated, quantitative ITC characterization of biomolecular interactions. The approach integrates microfabricated differential calorimetric sensors with microfluidic titration. Biomolecules and reagents are introduced at each of a series of molar ratios, mixed, and allowed to react. The reaction thermal power is differentially measured, and used to determine the thermodynamic profile of the biomolecular interactions. Implemented in a microdevice featuring thermally isolated, well-defined reaction volumes with minimized fluid evaporation as well as highly sensitive thermoelectric sensing, the approach enables accurate and quantitative ITC measurements of protein–ligand interactions under different isothermal conditions. Using the approach, we demonstrate ITC characterization of the binding of 18-Crown-6 with barium chloride, and the binding of ribonuclease A with cytidine 2′-monophosphate within reaction volumes of approximately 0.7µL and at concentrations down to 2mM. For each binding system, the ITC measurements were completed with considerably reduced analysis times and material consumption, and yielded a complete thermodynamic profile of the molecular interaction in agreement with published data. This demonstrates the potential usefulness of our approach for biomolecular characterization in biomedical applications.

Application of differential scanning calorimetry to measure the differential binding of ions, water and protons in the unfolding of DNA molecules

BackgroundThe overall stability of DNA molecules globally depends on base-pair stacking, base-pairing, polyelectrolyte effect and hydration contributions. In order to understand how they carry out their biological roles, it is essential to have a complete physical description of how the folding of nucleic acids takes place, including their ion and water binding. Scope of reviewTo investigate the role of ions, water and protons in the stability and melting behavior of DNA structures, we report here an experimental approach i.e., mainly differential scanning calorimetry (DSC), to determine linking numbers: the differential binding of ions (Δnion), water (ΔnW) and protons (ΔnH+) in the helix–coil transition of DNA molecules. General significanceWe use DSC and temperature-dependent UV spectroscopic techniques to measure the differential binding of ions, water, and protons for the unfolding of a variety of DNA molecules: salmon testes DNA (ST-DNA), one dodecamer, one undecamer and one decamer duplexes, nine hairpin loops, and two triplexes. These methods can be applied to any conformational transition of a biomolecule. Major conclusionsWe determined complete thermodynamic profiles, including all three linking numbers, for the unfolding of each molecule. The favorable folding of a DNA helix results from a favorable enthalpy-unfavorable entropy compensation. DSC thermograms and UV melts as a function of salt, osmolyte and proton concentrations yielded releases of ions and water. Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of both counterions and water molecules. In addition, the triplex with C+GC base triplets yielded an uptake of protons. Furthermore, the folding of a DNA duplex is accompanied by a lower uptake of ions and a similar uptake of four water molecules as the DNA helix gets shorter. In addition, the oligomer duplexes and hairpin thermodynamic data suggest ion and water binding depends on the DNA sequence rather than DNA composition. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.

Study on the interaction of the toxic food additive carmoisine with serum albumins: A microcalorimetric investigation

The interaction of the synthetic azo dye and food colorant carmoisine with human and bovine serum albumins was studied by microcalorimetric techniques. A complete thermodynamic profile of the interaction was obtained from isothermal titration calorimetry studies. The equilibrium constant of the complexation process was of the order of 106M−1 and the binding stoichiometry was found to be 1:1 with both the serum albumins. The binding was driven by negative standard molar enthalpy and positive standard molar entropy contributions. The binding affinity was lower at higher salt concentrations in both cases but the same was dominated by mostly non-electrostatic forces at all salt concentrations. The polyelectrolytic forces contributed only 5–8% of the total standard molar Gibbs energy change. The standard molar enthalpy change enhanced whereas the standard molar entropic contribution decreased with rise in temperature but they compensated each other to keep the standard molar Gibbs energy change almost invariant. The negative standard molar heat capacity values suggested the involvement of a significant hydrophobic contribution in the complexation process. Besides, enthalpy–entropy compensation phenomenon was also observed in both the systems. The thermal stability of the serum proteins was found to be remarkably enhanced on binding to carmoisine.

Thermodynamic and Structural Determinants of Differential Pdx1 Binding to Elements from the Insulin and IAPP Promoters

In adult mammals, the production of insulin and other peptide hormones, such as the islet amyloid polypeptide (IAPP), is limited to β-cells due to tissue-specific expression of a set of transcription factors, the best known of which is pancreatic duodenal homeobox protein 1 (Pdx1). Like many homeodomain transcription factors, Pdx1 binds to a core DNA recognition sequence containing the tetranucleotide 5′-TAAT-3′; its consensus recognition element is 5′-CTCTAAT(T/G)AG-3′. Currently, a complete thermodynamic profile of Pdx1 binding to near-consensus and native promoter sequences has not been established, obscuring the mechanism of target site selection by this critical transcription factor. Strikingly, while Pdx1 responsive elements in the human insulin promoter conform to the pentanucleotide 5′-CTAAT-3′ sequence, the Pdx1 responsive elements in the human iapp promoter all contain a substitution to 5′-TTAAT-3′. The crystal structure of Pdx1 bound to the consensus nucleotide sequence does not explain how Pdx1 identifies this natural variation, if it does at all. Here we report a combination of isothermal calorimetric titrations, NMR spectroscopy, and extensive multi-microsecond molecular dynamics calculations of Pdx1 that define its interactions with a panel of natural promoter elements and consensus-derived sequences. Our results show a small preference of Pdx1 for a C base 5′ relative to the core TAAT promoter element. Molecular mechanics calculations, corroborated by experimental NMR data, lead to a rational explanation for sequence discrimination at this position. Taken together, our results suggest a molecular mechanism for differential Pdx1 affinity to elements from the insulin and iapp promoter sequences.

83 Application of differential scanning calorimetry to measure the differential binding of ions, water, and protons in the unfolding of DNA molecules

The overall stability of DNA molecules globally depends on base-pair stacking, base pairing, polyelectrolyte effect, and hydration contributions. In order to improve our understanding of the role of ions, water, and protons in the stability and melting behavior of DNA structures, we report an experimental approach to determine the differential binding of ions (Δn ion), water (Δn W), and protons (Δn H+) in the helix-coil transition of DNA molecules. A combination of differential scanning calorimetry (DSC) and temperature-dependent UV and CD spectroscopic techniques to investigate the unfolding of a variety of DNA molecules: S.T. DNA, two dodecamers, one undecamer, nine short hairpins as a function of the GC content of their stem, and two triplexes. We determine complete thermodynamic profiles, including all the three linking numbers, for the unfolding of each molecule. The CD spectra indicated that all molecules adopted the B-conformation at low temperatures. Thermodynamic profiles obtained from the DSC curves indicate that the favorable folding of each molecule results from the typical compensation of favorable enthalpy and unfavorable entropy contributions, and negligible heat capacity effects. UV and DSC melting curves as a function of salt, osmolyte, and proton concentrations yielded releases of ions, water, and protons (for the triplex with C+GC base triplets). Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of water and counterions. The thermodynamic data will be discussed in terms of the effects of DNA length, loop contributions and type of water molecules.

84 PPC measurements of unfolding volumes of DNA stem-loop motifs

One focus of our research is to further our understanding of the physico-chemical properties of non-canonical nucleic acid structures. In this work, DNA hairpins are used to mimic a common motif present in RNA, i.e. a stem-loop motif with a bulge or internal loop in their stem. Specifically, we used a combination of temperature-dependent UV spectroscopy, differential scanning (DSC), and pressure perturbation (PPC) calorimetric techniques to determine complete thermodynamic profiles for the helix–coil transitions of two sets of hairpins with 5′–3′ sequences: d(GCGCT n GTAACT5GTTACGCGC) and d(GCGCT n GTAACT5GTTACT n GCGC). “T n ” is a variable loop of thymines, n = 1, 3 or 5; and “T5” is an end-loop of five thymines. Unfolding curves show monophasic transitions with TMs independent of strand concentration, confirming their intramolecular formation. DSC thermodynamic profiles indicate that the favorable folding of each hairpin results from the typical compensation of favorable enthalpy and unfavorable entropy contributions, while the DSC curves as a function of salt concentration yielded an uptake of cations and negative heat capacity effects. PPC melting curves yielded positive folding volumes ranging 12–31 cm3/mol, corresponding to releases of water molecules; in contrast, an uptake of water (ranging from 32 to 63 mol of H2O/mol) is observed from osmotic stress experiments using ethylene glycol as the osmolyte. Overall, the increase in the size of the variable bulge or internal-loop yielded lower TMs and slightly more favorable enthalpies, corresponding to less favorable free energy contributions of ∼0.7 kcal/mol per thymine residue. The volume measurements will be correlated with the unfolding entropies and discussed in terms of the type of water that is hydrating these stem-loop motifs structures.