Gorazd Vesnaver

Thermodynamic investigation of the effect of octanol–water mutual miscibility on the partitioning and solubility of some guanine derivatives

The effects of mutual saturation of octanol and water on solute partitioning and solubility have been examined. The partition coefficients and solubilities in octanol, water and mutually saturated octanol–water phases were determined for eight antivirus guanine derivatives. Using the solubility data, enthalpies of fusion (ΔfusH) and melting temperatures (Tm), determined by differential scanning calorimetry (DSC), the activity coefficients of the solutes in the aqueous and organic phases were calculated. From these results the intermolecular interactions among the solute and solvent molecules in the aqueous and octanol phases were estimated. It has been shown that the mutual saturation of the octanol and aqueous phases plays an important role in the partitioning of semipolar substances, such as guanine derivatives.

Studies by UV spectroscopy of thermal denaturation of β-lactoglobulin in urea and alkylurea solutions

The thermal denaturation of β-lactoglobulin in aqueous solutions at three different pHs and in aqueous solutions of urea, methyl-, N,N′-dimethyl- and ethylurea was studied by UV spectroscopy. The UV-melting curves were analyzed on the basis of two-state approximation to obtain the apparent equilibrium constant, Kapp, and the apparent standard enthalpy of transition, ΔHapp0, for protein unfolding as a function of temperature. From Kapp, calculations of ΔGapp0, as functions of temperature around transition temperature, T12, in urea and alkylurea solutions and different buffer solutions have been carried out. An increase in the observed transition temperature, T12, and the corresponding transition enthalpies, ΔHapp0, with decreasing pH or denaturant concentration indicate increased stability of protein in these conditions. However, comparison of ΔHapp0 with ΔHcal shows that the thermal transition of β-lactoglobulin in aqueous urea and alkylurea solutions is not a two-state process. It has also been observed that urea and all alkylureas cause a red shift in the absorbance spectrum of β-lactoglobulin which increases with increasing denaturant concentration and decreasing pH. A similar increase in red shift of β-lactoglobulin absorbance spectrum has also been observed with increasing temperature.

Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes.

Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation.

Driving Forces of Gyrase Recognition by the Addiction Toxin CcdB

Gyrase, an essential bacterial topoisomerase, is the target of several antibiotics (e.g. quinolones) as well as of bacterial toxin CcdB. This toxin, encoded by Escherichia coli toxin-antitoxin module ccd, poisons gyrase by causing inhibition of both transcription and replication. Because the molecular driving forces of gyrase unfolding and CcdB-gyrase binding were unknown, the nature of the CcdB-gyrase recognition remained elusive. Therefore, we performed a detailed thermodynamic analysis of CcdB binding to several fragments of gyrase A subunit (GyrA) that contain the CcdB-binding site. Binding of CcdB to the shorter fragments was studied directly by isothermal titration calorimetry. Its binding to the longer GyrA59 fragment in solution is kinetically limited and was therefore investigated via urea induced unfolding of the GyrA59-CcdB complex and unbound GyrA59 and CcdB, monitored by circular dichroism spectroscopy. Model analysis of experimental data, in combination with the relevant structural information, indicates that CcdB binding to gyrase is an enthalpic process driven mainly by specific interactions between CcdB and the highly stable dimerization domain of the GyrA. The dissection of binding energetics indicates that CcdB-gyrase recognition is accompanied by opening of the tower and catalytic domain of GyrA. Such extensive structural rearrangements appear to be crucial driving forces for the functioning of the ccd toxin-antitoxin module.

What drives the binding of minor groove-directed ligands to DNA hairpins?

Understanding the molecular basis of ligand–DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule.

INTERACTION OF 3-ALKYLPYRIDINIUM POLYMERS FROM THE SEA SPONGE RENIERA SARAI WITH INSECT ACETYLCHOLINESTERASE

Abstract3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS–PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS–PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.

Diverse Polymorphism of G-Quadruplexes as a Kinetic Phenomenon

Knowledge of forces that drive conformational transitions of G-quadruplexes is crucial for understanding the molecular basis of several key cellular processes. It can only be acquired by combining structural, thermodynamic and kinetic information. Existing biophysical and structural evidences on polymorphism of intermolecular G-quadruplexes have shown that the formation of a number of these structures is a kinetically controlled process. Reported kinetic models that have been used to describe the association of single strands into quadruplex structures seem to be inappropriate since the corresponding model-predicted activation energies turn out to be negative. By contrast, we propose here a novel kinetic model that successfully describes experimentally monitored folding/unfolding transitions of G-quadruplexes and gives positive activation energies for all elementary steps, including those describing association of two single strands into bimolecular quadruplex structures. It is based on a combined thermodynamic and kinetic investigation of polymorphic behavior of bimolecular G-quadruplexes formed from d(G4T4G4) and d(G4T4G3) strands in the presence of Na(+) ions, monitored by spectroscopic (UV, CD) and calorimetric (DSC) techniques. According to our experiment and model analysis the topology of the measured G-quadruplexes is clearly flexible with the conformational forms that respond to the rate of temperature change at which global unfolding/folding transitions occur.

Fluorescence studies of the effect of pH, guanidine hydrochloride and urea on equinatoxin II conformation

The solvent denaturation of equinatoxin II (EqTxII) in aqueous solutions of urea, guanidine hydrochloride (Gu-HCl) and at various pH values was examined by monitoring changes in the protein intrinsic emission fluorescence spectra and in the fluorescence spectra of the added external probe ANS. It has been observed that EqTxII denaturation is reflected in a strong red shift of intrinsic fluorescence emission maxima accompanied by a simultaneous decrease in fluorescence intensity and that guanidine hydrochloride is significantly more powerful denaturant than urea or changing of pH. Comparison of intrinsic fluorescence spectra of EqTxII denatured by one of the three denaturing agents has shown that the fully denatured states of the protein in Gu-HCl and urea are similar and substantially different from those induced by changing of pH. Furthermore, according to the measurements of the ANS-fluorescence in EqTxII solutions as a function of pH the protein exists at pH values below 2.0 in an acid-denatured compact state.

Thermodynamics of denaturation of alpha-chymotrypsinogen A in aqueous urea and alkylurea solutions.

The effects of pH, urea, and alkylureas on the thermal stability ofα-chymotrypsinogen A (α-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition ofα-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25°C were calculated. Comparison of these values shows that at 25°C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein molecules. At pH lower than 2.0,α-ctg A still exists in a very stable form, probably the acid-denatured form (A-form).

Model-Based Thermodynamic Analysis of Reversible Unfolding Processes

Folding and unfolding of many biological macromolecules can be characterized thermodynamically, yielding a wealth of information about the stability of various conformations and the interactions that hold them together. The relevant thermodynamic parameters are usually obtained by employing spectroscopic and/or calorimetric techniques and fitting an appropriate thermodynamic model to the experimental data. In this work, we compare the traditional approach of fitting the thermodynamic model to experimental data obtained from each experiment individually and the global approach of simultaneously fitting the model to all available data from different experiments. On the basis of several specific examples of DNA and protein unfolding, we demonstrate that piece-by-piece verification of the proposed thermodynamic model using individual fits is frequently inappropriate and can result in an incorrect mechanism and thermodynamics of the studied unfolding process. We find that while the two approaches are complementary in some aspects of analysis global fitting is essential for the appropriate selection and critical evaluation of the model mechanism. Only a good global fit thus gives us confidence that the obtained thermodynamic parameters of unfolding have real physical meaning.