Application of isothermal titration calorimetry in evaluation of protein–nanoparticle interactions

Nanoparticles (NPs) offer a number of advantages over small organic molecules for controlling protein behaviour inside the cell. Protein binding to the surface of NPs depends on their surface characteristics, composition and method of preparation (Mandal et al. in J Hazard Mater 248–249:238–245, 2013). It is important to understand the binding affinities, stoichiometries and thermodynamical parameters of NP–protein interactions in order to see which interaction will have toxic and hazardous consequences and thus to prevent it. On the other side, because proteins are on the brink of stability, they may experience interactions with some types of NPs that are strong enough to cause denaturation or significantly change their conformations with concomitant loss of their biological function. Structural changes in the protein may cause exposure of new antigenic sites, “cryptic” peptide epitopes, potentially triggering an immune response which can promote autoimmune disease (Treuel et al. in ACS Nano 8(1):503–513, 2014). Mechanistic details of protein structural changes at NP surface have still remained elusive. Understanding the formation and persistence of the protein corona is critical issue; however, there are no many analytical methods which could provide detailed information about the NP–protein interaction characteristics and about protein structural changes caused by interactions with nanoparticles. The article reviews recent studies in NP–protein interactions research and application of isothermal titration calorimetry (ITC) in this research. The study of protein structural changes upon adsorption on nanoparticle surface and application of ITC in these studies is emphasized. The data illustrate that ITC is a versatile tool for evaluation of interactions between NPs and proteins. When coupled with other analytical methods, it is important analytical tool for monitoring conformational changes in proteins

Differential scanning calorimetry to investigate G-quadruplexes structural stability

Differential Scanning Calorimetry (DSC) is a straightforward methodology to characterize the energetics of thermally-induced transitions of DNA and other biological macromolecules. Therefore, DSC has been used to study the thermodynamic stability of several nucleic acids structures. G-quadruplexes are among the most important non-canonical nucleic acid architectures that are receiving great consideration. This article reports examples on the contribution of DSC to the knowledge of G-quadruplex structures. The selected case studies show the potential of this method in investigating the structure stability of G-quadruplex forming nucleic acids, and in providing information on their structural complexity. Indeed, DSC can determine thermodynamic parameters of G-quadruplex folding/unfolding processes, but it can also be useful to reveal the formation of multiple conformations or the presence of intermediate states along the unfolding pathway, and to evaluate the impact of chemical modifications on their structural stability. This article aims to show that DSC is an important complementary methodology to structural techniques, such as NMR and X-ray crystallography, in the study of G-quadruplex forming nucleic acids

THERMODYNAMICS OF INTERACTION BETWEEN RECOMBINANT HUMAN LYSOSOMAL alfa-GLUCOSIDASE AND PHARMACOLOGICAL CHAPERONES

Background : Glycogen storage disease type II or Pompe disease is a rare inherited metabolic. Pompe disease is caused by mutations in the gene that encodes the lysosomal hydrolase acid a-glucosidase (GAA). GAA deficiency results in lysosomal glycogen accumulation and cellular dysfunction in multiple tissues, particularly skeletal and cardiac muscle . Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA; Myozyme, Genzyme Corporation, Cambridge, MA) is the only approved treatment for Pompe disease. Recently, it has been demonstrated that pharmacological chaperones, small molecules that specifically bind and stabilize target proteins, can also enhance the trafficking and cellular activity of a number of different wild-type and mutant forms of lysosomal hydrolases. A general strategy for lysosomal storage disorder is also to search for small pharmacological chaperones that are able to bind the active site of mutant enzymes and to facilitate the folding and transport process. These molecules have the ability to improve the trafficking of the protein between the endoplasmic reticulum (ER) and the lysosome. Various inhibitors derived from deoxynojirimycin (DNJ) have been evaluated as pharmacological chaperones, in different lysosomal storage disorders . Methods : We examined the binding affinity of pharmacological chaperones such as 1-deoxynojirimycin (DNJ) and N-acetylcysteine (NAC) to the rhGAA, Myozyme by Isothermal Titration Calorimetry. We studied thermal stability of the enzyme in the absence and presence of ligands at neutral and acidic pH by Differential Scanning Calorimetry Results : ITC results allowed a complete thermodynamic characterization of the interaction between the studied small ligand and the enzyme while the DSC results gave important information on the enzyme conformational stability as a function of pH

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. © 2012 American Chemical Society