Concetta Giancola

DSC studies on bovine serum albumin denaturation Effects of ionic strength and SDS concentration

This work analyzed the thermal denaturation process of defatted bovine serum albumin (BSA). DSC measurements were performed on changing the pH, the ionic strength and the sodium dodecyl sulfate (SDS) concentration. These data have been compared with those previously obtained by us and other authors. The purpose of these measurements was to study the correlation between the three-dimensional organization of BSA native protein structure and its thermodynamic stability and to clarify the non-covalent interactions between the globular proteins and amphipathic molecules. These measurements have shown that the thermal denaturation is always irreversible regardless of pH, ionic strength and SDS concentration. The nature of the irreversible process superimposed on the protein unfolding is discussed. The strong stabilizing effect of NaCl on the BSA native structure has been found for the range 0-1.0 M. It is worth noting that the calorimetric curves, confined to the pH region studied, could not be represented by a two-state transition model; they were deconvoluted as the sum of two independent two-state transitions. These transitions were correlated to the domain structure of BSA. Sodium dodecyl sulfate has a net stabilizing effect up to a molar ratio of 10:1 (ligand to protein). In this range of concentrations the presence of SDS cause a biphasic profile of excess heat capacity. A simple thermodynamic model was developed in attempt to reproduce the experimental DSC profiles and collect information regarding the binding equilibrium of SDS.

Thrombin–aptamer recognition: a revealed ambiguity

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human α-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5′-5′ polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin–mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin–aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.

A new modified thrombin binding aptamer containing a 5′–5′ inversion of polarity site

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5′–5′ inversion of polarity site, namely d(3′GGT5′-5′TGGTGTGGTTGG3′), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(3′GGT5′-5′TGGTGTGGTTGG3′) is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4°C higher than the corresponding unmodified TBA. d(3′GGT5′-5′TGGTGTGGTTGG3′) continues to display an anticoagulant activity, even if decreased with respect to the TBA.

Theseus: A new software package for the handling and analysis of thermal denaturation data of biological macromolecules

A new software package (THESEUS) has been assembled for the analysis of the DSC data, Concerning the thermal denaturation of biological macromolecules. The system is useful to obtain accurate physico-chemical information, bypassing the casual and systematic errors, very common in these experiments. It can also be used for handling data from other instruments and methodologies giving thermodynamic, spectroscopic or other kind of data as a function of temperature. Because many of the researches in this field are of exploratory nature and continuously new unfolding mechanisms are described or hypothesized in the current literature, we have written and assembled this powerful and flexible program of general applicability, in order to put the operator in a position to control each step of the calculation procedure and use his own experience for choosing the better way to solve unexpected problems.ZusammenfassungBei der Analyse von DSC-Daten der thermischen Zersetzung von biologischen Makromolekülen wurde ein neues Softwarepaket (THESEUS) angewendet. Unter Umgehung der in derartigen Experimenten oft vorkommenden gelegentlichen und systematischen Fehler eignet sich dieses System, um genaue Physikalisch-chemische Informationen zu erhalten. Außerdem kann es zur Verarbeitung von Daten verwendet werden, welche von anderen Instrumenten und Methoden stammen, die thermodynamische, spektroskopische oder andere Daten als Funktion der Temperatur liefern. Da viele der Untersuchungen auf diesem Gebiet Forschungs-Charakter tragen und in der gegen-wärtigen Literatur ständig neue Dekonvolutionsmechanismen beschrieben oder angenommen werden, haben wir dieses leistungsstarke, flexible und allgemein anwendbare Programm geschrieben und umgesetzt, um den Operator in die Lage zu versetzen, jeden Phase des Rechenvorganges kontrollieren und seine eigene Erfahrung benutzen zu können, um den besten Weg zur Lösung unvorhergesehener Probleme zu finden.

Applications of Isothermal Titration Calorimetry in Biophysical Studies of G-quadruplexes

G-quadruplexes are higher-order nucleic acids structures formed by G-rich sequences that are stabilized by tetrads of hydrogen-bonded guanine bases. Recently, there has been growing interest in the study of G-quadruplexes because of their possible involvement in many biological processes. Isothermal titration calorimetry (ITC) has been proven to be a useful tool to study the energetic aspects of G-quadruplex interactions. Particularly, ITC has been applied many times to determine the thermodynamic properties of drug-quadruplex interactions to screening among various drugs and to address drug design. In the present review, we will focus on the ITC studies of G-quadruplex structures and their interaction with proteins and drugs and the most significant results will be discussed.

Targeting DNA quadruplexes with distamycin A and its derivatives: An ITC and NMR study

The use of small molecules that bind and stabilize G-quadruplex structures is emerging as a promising way to inhibit telomerase activity in tumor cells. In this paper, isothermal titration calorimetry (ITC) and 1H NMR studies have been conducted to examine the binding of distamycin A and its two carbamoyl derivatives (compounds 1 and 2) to the target [d(TGGGGT)]4 and d[AG3(T2AG3)3] quadruplexes from the Tetrahymena and human telomeres, respectively. The interactions were examined using two different buffered solutions containing either K+ or Na+ at a fixed ionic strength, to evaluate any influence of the ions present in solution on the binding behaviour. Experiments reveal that distamycin A and compound 1 bind the investigated quadruplexes in both solution conditions; conversely, compound 2 appears to have a poor affinity in any case. Moreover, these studies indicate that the presence of different cations in solution affects the stoichiometry and thermodynamics of the interactions.

Selective Binding of Distamycin A Derivative to G-Quadruplex Structure [d(TGGGGT)](4).

Guanine-rich nucleic acid sequences can adopt G-quadruplex structures stabilized by layers of four Hoogsteen-paired guanine residues. Quadruplex-prone sequences are found in many regions of human genome and in the telomeres of all eukaryotic organisms. Since small molecules that target G-quadruplexes have been found to be effective telomerase inhibitors, the identification of new specific ligands for G-quadruplexes is emerging as a promising approach to develop new anticancer drugs. Distamycin A is known to bind to AT-rich sequences of duplex DNA, but it has recently been shown to interact also with G-quadruplexes. Here, isothermal titration calorimetry (ITC) and NMR techniques have been employed to characterize the interaction between a dicationic derivative of distamycin A (compound 1) and the [d(TGGGGT)]4 quadruplex. Additionally, to compare the binding behaviour of netropsin and compound 1 to the same target, a calometric study of the interaction between netropsin and [d(TGGGGT)]4 has been performed. Experiments show that netropsin and compound 1 are able to bind to [d(TGGGGT)]4 with good affinity and comparable thermodynamic profiles. In both cases the interactions are entropically driven processes with a small favourable enthalpic contribution. Interestingly, the structural modifications of compound 1 decrease the affinity of the ligand toward the duplex, enhancing the selectivity.

The interaction of the Escherichia coli protein SlyD with nickel ions illuminates the mechanism of regulation of its peptidyl-prolyl isomerase activity

The sensitive to lysis D (SlyD) protein from Escherichia coli is related to the FK506‐binding protein family, and it harbours both peptidyl‐prolyl cis–trans isomerase (PPIase) and chaperone‐like activity, preventing aggregation and promoting the correct folding of other proteins. Whereas a functional role of SlyD as a protein‐folding catalyst in vivo remains unclear, SlyD has been shown to be an essential component for [Ni–Fe]‐hydrogenase metallocentre assembly in bacteria. Interestingly, the isomerase activity of SlyD is uniquely modulated by nickel ions, which possibly regulate its functions in response to external stimuli. In this work, we investigated the solution structure of SlyD and its interaction with nickel ions, enabling us to gain insights into the molecular mechanism of this regulation. We have revealed that the PPIase module of SlyD contains an additional C‐terminal α‐helix packed against the catalytic site of the domain; unexpectedly, our results show that the interaction of SlyD with nickel ions entails participation of the novel structural features of the PPIase domain, eliciting structural alterations of the catalytic pocket. We suggest that such conformational rearrangements upon metal binding underlie the ability of nickel ions to regulate the isomerase activity of SlyD.

Energetics of Quadruplex-Drug Recognition in Anticancer Therapy

Immortality of tumour cells is strictly correlated to telomerase activity. Telomerase is overexpressed in about 85% of tumour cells and maintains telomere length contributing to cell immortalisation, whereas in somatic cells telomeres progressively shorten until cell death occurs by apoptosis. Different drugs can promote telomeric G-rich overhangs which fold into quadruplex structures that inhibit telomerase activity. Detailed studies on drug-quadruplex complexes are essential to understand quadruplex recognition and address drug design. This review will discuss the energetic aspects of quadruplex-drug interactions with a particular attention to physico-chemical methodologies.

Shooting for Selective Druglike G‑Quadruplex Binders: Evidence for Telomeric DNA Damage and Tumor Cell Death

Targeting of DNA secondary structures, such as G-quadruplexes, is now considered an appealing opportunity for drug intervention in anticancer therapy. So far, efforts made in the discovery of chemotypes able to target G-quadruplexes mainly succeeded in the identification of a number of polyaromatic compounds featuring end-stacking binding properties. Against this general trend, we were persuaded that the G-quadruplex grooves can recognize molecular entities with better drug-like and selectivity properties. From this idea, a set of small molecules was identified and the structural features responsible for G-quadruplex recognition were delineated. These compounds were demonstrated to have enhanced affinity and selectivity for the G-quadruplex over the duplex structure. Their ability to induce selective DNA damage at telomeric level and to induction of apoptosis and senescence on tumor cells is herein experimentally proven.