Characterization of a G-quadruplex from hepatitis B virus and its stabilization by binding TMPyP4, BRACO19 and PhenDC3.

Specific guanine rich nucleic acid sequences can form non-canonical structures, like the four stranded G-quadruplex (GQ). We studied the GQ-forming sequence (named HepB) found in the genome of the hepatitis B virus. Fluorescence-, infrared- and CD-spectroscopy were used. HepB shows a hybrid form in presence of K+, but Na+, Li+, and Rb+ induce parallel structure. Higher concentrations of metal ions increase the unfolding temperature, which was explained by a short thermodynamic calculation. Temperature stability of the GQ structure was determined for all these ions. Na+ has stronger stabilizing effect on HepB than K+, which is highly unusual. The transition temperatures were 56.6, 53.8, 58.5 and 54.4 °C for Na+, K+, Li+, and Rb+ respectively. Binding constants for Na+ and K+ were 10.2 mM and 7.1 mM respectively. Study of three ligands designed in cancer research for GQ targeting (TMPyP4, BRACO19 and PhenDC3) showed unequivocally their binding to HepB. Binding was proven by the increased stability of the bound form. The stabilization was higher than 20 °C for TMPyP4 and PhenDC3, while it was considerably lower for BRACO19. These results might have medical importance in the fight against the hepatitis B virus.

Pressure Perturbation Studies of Noncanonical Viral Nucleic Acid Structures.

G-quadruplexes are noncanonical structures formed by guanine-rich sequences of the genome. They are found in crucial loci of the human genome, they take part in the regulation of important processes like cell proliferation and cell death. Much less is known about the subjects of this work, the viral G-quadruplexes. We have chosen three potentially G-quadruplex-forming sequences of hepatitis B. We measured the stability and the thermodynamic parameters of these quadruplexes. We also investigated the potential stabilization of these G-quadruplexes by binding a special ligand that was originally developed for cancer therapy. Fluorescence and infrared spectroscopic measurements were performed over wide temperature and pressure ranges. Our experiments indicate the small unfolding volume change of all three oligos. We found a difference between the unfolding of the 2-quartet and the 3-quartet G-quadruplexes. All three G-quadruplexes were stabilized by TMPyP4, which is a cationic porphyrin developed for stabilizing the human telomere.

Crosslinked Hyaluronic Acid Gels with Blood-Derived Protein Components for Soft Tissue Regeneration.

Hyaluronic acid (HA) is an ideal initial material for preparing hydrogels, which may be used as scaffolds in soft tissue engineering based on their advantageous physical and biological properties. In this study, two crosslinking agents, divinyl sulfone (DVS) and butanediol diglycidyl ether, were used to investigate their effect on the properties of HA hydrogels. As HA hydrogels alone do not promote cell adhesion on the scaffold, fibrin and serum from platelet-rich fibrin (SPRF) were combined with the scaffold; the aim was to create a material intended to be used as soft tissue implant that facilitates new tissue formation, and degrades over time. The chemical changes were characterized and cell attachment capacity of the protein-containing gels was examined using human mesenchymal stem cells, and viability was assessed using live-dead staining. Fourier-transform infrared measurements revealed that linking fibrin into the gel was more effective than linking SPRF. The scaffolds were found to be able to support cell adherence onto the hydrogels, and the best result was achieved when HA was crosslinked with DVS and contained fibrin. The most promising derivative, 5% DVS-crosslinked fibrin-containing hydrogel, was injected subcutaneously into C57BL/6 mice for 12 weeks. The scaffold was proven to be biocompatible, remodeling, and vascularization occurred, while shape and integrity were maintained. Impact statement Fibrin was combined with crosslinked hyaluronic acid (HA) for regenerative application, the structure of the combination of crosslinked HA with blood-derived protein was analyzed and effective coating was proven. It was observed that the fibrin content led to better mesenchymal stem cell attachment in vitro. The compositions showed biocompatibility, connective tissue and vascularization took place when implanted in vivo. Thus, a biocompatible, injectable gel was produced, which is a potential candidate for soft tissue implantation.

Negative volume changes of human G-quadruplexes at unfolding.

G-quadruplexes are tetrahelical structures. They are important targets for anti-cancer drugs, since they are situated at crucial positions within the genome. We studied the volumetric properties of the unfolding of three G-quadruplexes in the presence of potassium ion. The unfolding volume changes were determined using high-pressure fluorescence spectroscopy. The c-MYC, KIT, and VEGF sequences unfold with the transition volume of -17, -6 and -18 cm3/mol, respectively. The small magnitude of the unfolding volume of KIT could be explained by its unique structure and the lower amount of void volume. Since the cell interior is highly crowded, the available volume is restricted. Therefore the volumetric changes during the conformational transformations gain biological importance.

Revealing unfolding steps and volume changes of human telomeric i-motif DNA.

Cytosine rich DNA sequences can fold into a non-canonical four stranded intercalated i-motif structure. We investigated Htel-iM, an i-motif structure of the human telomeric DNA region that plays an important role in cell division, cancer diseases and aging. A high pressure up to 1 Gpa was applied to reveal the volumetric changes during unfolding. Thermal transitions at different pressures were followed by infrared and fluorescence spectroscopy. We demonstrated that Htel-iM unfolds in two steps. First the outer hydrogen bonds break in which C(2)[double bond, length as m-dash]O(2) and the N(4)H2 amino group is involved, subsequently the hydrogen bond involving N(3)+ becomes broken. Htel-iM was destabilized by pressure and unfolded with a negative volume change.

Structural and nanomechanical comparison of epitaxially and solution-grown amyloid ß25-35 fibrils.

Aß25-35, the fibril-forming, biologically active toxic fragment of the full-length amyloid ß-peptide also forms fibrils on mica by an epitaxial assembly mechanism. Here we investigated, by using atomic force microscopy, nanomechanical manipulation and FTIR spectroscopy, whether the epitaxially grown fibrils display structural and mechanical features similar to the ones evolving under equilibrium conditions in bulk solution. Unlike epitaxially grown fibrils, solution-grown fibrils displayed a heterogeneous morphology and an apparently helical structure. While fibril assembly in solution occurred on a time scale of hours, it appeared within a few minutes on mica surface fibrils. Both types of fibrils showed a similar plateau-like nanomechanical response characterized by the appearance of force staircases. The IR spectra of both fibril types contained an intense peak between 1620 and 1640 cm(-1), indicating that ß-sheets dominate their structure. A shift in the amide I band towards greater wave numbers in epitaxially assembled fibrils suggests that their structure is less compact than that of solution-grown fibrils. Thus, equilibrium conditions are required for a full structural compaction. Epitaxial Aß25-35 fibril assembly, while significantly accelerated, may trap the fibrils in less compact configurations. Considering that under in vivo conditions the assembly of amyloid fibrils is influenced by the presence of extracellular matrix components, the ultimate fibril structure is likely to be influenced by the features of underlying matrix elements.

High pressure effects on allergen food proteins.

There are several proteins, which can cause allergic reaction if they are inhaled or ingested. Our everyday food can also contain such proteins. Food allergy is an IgE-mediated immune disorder, a growing health problem of great public concern. High pressure is known to affect the structure of proteins; typically few hundred MPa pressure can lead to denaturation. That is why several trials have been performed to alter the structure of the allergen proteins by high pressure, in order to reduce its allergenicity. Studies have been performed both on simple protein solutions and on complex food systems. Here we review those allergens which have been investigated under or after high pressure treatment by methods capable of detecting changes in the secondary and tertiary structure of the proteins. We focus on those allergenic proteins, whose structural changes were investigated by spectroscopic methods under pressure in correlation with the observed allergenicity (IgE binding) changes. According to this criterion we selected the following allergen proteins: Mal d 1 and Mal d 3 (apple), Bos d 5 (milk), Dau c 1 (carrot), Gal d 2 (egg), Ara h 2 and Ara h 6 (peanut), and Gad m 1 (cod).

Different pressure-temperature behavior of the structured and unstructured regions of titin.

Contrary to the classical view, according to which all proteins adopt a specific folded conformation necessary for their function, intrinsically unstructured proteins (IUPs) display random-coil-like conformation under physiological conditions. We compared the structured and unstructured domains from titin, a giant protein responsible for striated-muscle elasticity. A 171-residue-long fragment (polyE) of the disordered PEVK domain, and an Ig domain (I27) with ordered structure were investigated. FTIR (Fourier transform infrared) and fluorescence spectroscopy combined with a diamond anvil cell were used for investigation of the secondary structures under wide range of pressure and temperature. PolyE preserves its disordered characteristics across the entire range of investigated pressure (0-16kbar), temperature (0-100°C), pD (3-10.5) and different solvent conditions. The detailed temperature-pressure phase diagram of titin I27 was determined. At 30°C, increasing pressure unfolds titin I27 in one step at 10.5kbar. Increasing temperature at atmospheric pressure results in two transitions. At 50°C the secondary structure is loosened and the protein transforms into a molten-globule state. At 65°C the protein completely unfolds. Unfolding is followed by aggregation at ambient pressure. Moderate pressures (>2kbar), however, can prevent the protein from aggregation. Our experiments in wide range of physical parameters revealed four different structures for I27, while the unstructured character of the PEVK fragment is insensitive to these parameters.

Pressure-temperature stability, Ca2+ binding, and pressure-temperature phase diagram of cod parvalbumin: Gad m 1.

Fish allergy is associated with IgE-mediated hypersensitivity reactions to parvalbumins, which are small calcium-binding muscle proteins and represent the major and sole allergens for 95% of fish-allergic patients. We performed Fourier transform infrared and tryptophan fluorescence spectroscopy to explore the pressure-temperature (p-T) phase diagram of cod parvalbumin (Gad m 1) and to elucidate possible new ways of pressure-temperature inactivation of this food allergen. Besides the secondary structure of the protein, the Ca(2+) binding to aspartic and glutamic acid residues was detected. The phase diagram was found to be quite complex, containing partially unfolded and molten globule states. The Ca(2+) ions were essential for the formation of the native structure. A molten globule conformation appears at 50 °C and atmospheric pressure, which converts into an unordered aggregated state at 75 °C. At >200 MPa, only heat unfolding, but no aggregation, was observed. A pressure of 500 MPa leads to a partially unfolded state at 27 °C. The complete pressure unfolding could only be reached at an elevated temperature (40 °C) and pressure (1.14 GPa). A strong correlation was found between Ca(2+) binding and the protein conformation. The partially unfolded state was reversibly refolded. The completely unfolded molecule, however, from which Ca(2+) was released, could not refold. The heat-unfolded protein was trapped either in the aggregated state or in the molten globule state without aggregation at elevated pressures. The heat-treated and the combined heat- and pressure-treated protein samples were tested with sera of allergic patients, but no change in allergenicity was found.

Pressure and temperature stability of the main apple allergen Mal d1.

High-pressure Fourier-transform infrared (FTIR) spectroscopy was used to determine the pressure and temperature stability of Mal d1. This study was triggered by contradictory results in the literature regarding the success of pressure treatment in the destruction of the allergen. The protein unfolded at 55°C when heated at normal atmospheric pressure. We also studied the effect exerted on pressure stability by environmental factors, which can be important for the stability of the protein in the apple. The pressure unfolding was measured under different pD conditions, and the effect of sugar mixture similar to that of the apple and the effect of ionic strength were also studied. In all cases the allergen unfolded with a transition midpoint in the range of 150-250 MPa. Unfolding was irreversible and was followed by aggregation of the unfolded protein. Lowering the pD destabilized the protein, while addition of sugar mixture and of KCl had stabilizing effect.