Fabio De Matteis

Opposite Effects of Ca2+ and GTP Binding on Tissue Transglutaminase Tertiary Structure

Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides. The protein activity is known to be finely tuned by Ca2+ and GTP binding. In this study we report the effects of these ligands on the enzyme structure, as revealed by circular dichroism, and steady-state and dynamic fluorescence measurements. We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure. In particular the metal ions were responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS). Unlike Ca2+, the nucleotide binding increased the protein dynamics, reducing its rotational correlation lifetime from 32 to 25 ns, preventing also the binding of ANS into the protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a three-state denaturation mechanism, involving an intermediate species with the characteristics of the so-called “molten globule” state. The effect of GTP binding (but not that of Ca2+) had an important consequence on the stability of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least ≈0.7 kcal/mol. Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP. These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein conformational change which, decreasing the accessibility of the protein matrix to the solvent, renders more difficult the exposure of the active site.

Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy.

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of α-helix 2 (residues 35–46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020–3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry37, 3028–3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.

From nanospheres to microribbons: Self-assembled Eosin Y doped PMMA nanoparticles as photonic crystals

A modified emulsion synthesis of poly(methylmethacrylate) (PMMA) with the Eosin Y (EY), commercial chromophore, yields dye doped polymeric nanoparticles (PMMA@EY). A systematic investigation on the experimental parameters (monomer and initiator concentration, reaction time and MMA/EY molar ratio) has been explored to modulate physico-chemical properties of the dye doped polymeric colloids. Spherical shaped particles, doped with EY (0.5-3.0 wt%; loading efficiency η = 11-15%), with controlled diameters in the range 240-510 nm, low dispersity and ζ-potential values in the range between -42 mV and -59 mV, have been synthesized and characterized by means of UV-Vis spectrometry, Dynamic Light Scattering (DLS), laser Doppler electrophoresis and Scanning Electron Microscopy (SEM). Microribbons based on PMMA@EY nanoparticles have been fabricated by room temperature self-assembly of aqueous colloidal suspension on highly wettable glass substrates. Surface chemical treatment assisted the formation of long (up to few centimeters) regular ribbons with rectangular section. Lateral size and height of the structures have been controlled by changing the suspension concentration and/or the deposition volume: the higher suspension concentration produces larger and thicker ribbons and the higher deposited volume produces thicker ribbons (up to 23 μm with 198 μL of a 3 wt% suspension). Moreover, a transition from a film-like to a ribbon-like growth has been observed with increasing nanoparticles concentration. Short range ordering and photonic crystal features have been maintained in the fluorescent ribbon microarchitecture, resulting in a self-assembled material with excellent potential for the development of mirror-less and random lasers.

Outstanding Poling Stability of a New Cross-Linked Nonlinear Optical (NLO) Material from a Low Molecular Weight Chromophore

In this paper we report the synthesis and characterization of a trihydroxylated nonlinear optical (NLO) azochromophore and its functionalization with 2,4-tolylendiisocyanate (TDI) to give an amorphous mixture of isomers that was used as a starting compound for the preparation of cross-linked electro-optic (EO) thin films. An unedited type of thermal cross-linking reaction was used, exploiting the reactivity of isocyanate groups themselves in the presence of N,N-dimethylacetamide, without the addition of any hydroxylated comonomer as usual in the preparation of polyurethanes. Thin films were prepared by spin coating and corona poled during thermal cross-linking. A d(33) value of 33 pm/V was obtained by second-harmonic generation (SHG) measurements on poled films, and an excellent stability of SHG signal was shown upon aging at 130 °C and during dynamic thermal stability measurements.

Hydrophilic silver nanoparticles with tunable optical properties: application for the detection of heavy metals in water

Due their excellent chemo-physical properties and ability to exhibit surface plasmon resonance, silver nanoparticles (AgNPs) have become a material of choice in various applications, such as nanosensors, electronic devices, nanobiotechnology and nanomedicine. In particular, from the environmental monitoring perspective, sensors based on silver nanoparticles are in great demand because of their antibacterial and inexpensive synthetic method. In the present study, we synthesized AgNPs in water phase using silver nitrate as precursor molecules, hydrophilic thiol (3-mercapto-1-propanesulfonic acid sodium salt, 3MPS) and sodium borohydride as capping and reducing agents, respectively. The AgNPs were characterized using techniques such as surface plasmon resonance (SPR) spectroscopy, dynamic light scattering (DLS), zeta potential (ζ-potential) measurements and scanning tunneling microscopy (STM). Further, to demonstrate the environmental application of our AgNPs, we also applied them for heavy metal sensing by detecting visible color modification due to SPR spectral changes. We found that these negatively charged AgNPs show good response to nickel (II) and presented good sensibility properties for the detection of low amount of ions in water in the working range of 1.0–0.1 ppm.

Dipolar Relaxation Times of Tryptophan and Tyrosine in Glycerol and in Proteins: A Direct Evaluation from Their Fluorescence Decays

The dipolar relaxation process induced around tryptophan, indole and tyrosine in viscous media, as well as in several single tryptophan-containing proteins (staphylococcal nuclease, ribonuclease T1, melittin and albumin), has been studied by dynamic fluorescence measurements. A new theoretical model has been developed, including the relaxation dynamics directly in the fluorescence decay function. The phase shift and demodulation data have been fitted with this new algorithm which allows to resolve the different relaxation times influencing the fluorophore excited state. These parameters are in a good agreement with those measured with the traditional time-resolved emission spectroscopy. The results indicate that indeed a correlation exists between the radiative rate change obtained with the new model and the temporal spectral shift reported in the literature. Finally, this new approach has also been extended to the case of superoxide dismutase and phosphofructokinase, allowing to measure the relaxation time even in proteins lacking a temporal spectral shift during the fluorphores lifetime.

Asymmetrical Distribution of Intrinsic Fluorescence Lifetimes in Proteins

Dynamic fluorescence measurements of proteins in solution are often interpreted in terms of continuous distributions of lifetimes, which reflect the intrinsic structural heterogeneity of these systems. In several cases a single Gaussian or Lorentzian symmetric distribution has been used to fit the data. In this paper we describe a new nonsymmetric Lorentzian function which contains three free parameters (the center, the left, and the right widths) like the double-discrete exponential model (the two lifetimes and one preexponential factor). Simulated data in the frequency domain have been used to compare the fits obtained with these different approaches, introducing a new parameter, ρ, which quantitatively measures the asymmetry of the distribution or the ratio of the two preexponential factors, in the continuous and discrete models, respectively. Real measurements of a mixture of independent fluorophores, as well as of protein fluorescence decays, have also been performed and analyzed in terms of the new asymmetric function. The data have also been fitted with traditional discrete methods (such as the two- and the three-exponential decay) and with another asymmetric function, namely, the skewed Gaussian distribution.

Order relaxation of a poled azo dye in a high Tg, fully aromatic polyimide

A guest-host polymer system with potential use in electro-optic devices is discussed. The polymer host is a high Tg, fully aromatic polyimide and the guest chromophore is disperse red 19. Relaxation mechanisms of polar order after electric field alignment procedure have been investigated by measuring the isothermal decay of the macroscopic nonlinear optical coefficient d333 at different temperatures below glass transition temperature (Tg), upon the removal of the poling electric field. All the decay curves can be fitted by a double exponential function. Below Tg, the slower relaxation time shows an Arrhenius temperature dependence. An extrapolation to room temperature allows to predict the time stability which results to be longer than 30years. In addition, absorption spectra measurements of the films were performed before and immediately after poling procedure to estimate independently the polar order of the dipoles through the decrease of the absorption coefficients.A guest-host polymer system with potential use in electro-optic devices is discussed. The polymer host is a high Tg, fully aromatic polyimide and the guest chromophore is disperse red 19. Relaxation mechanisms of polar order after electric field alignment procedure have been investigated by measuring the isothermal decay of the macroscopic nonlinear optical coefficient d333 at different temperatures below glass transition temperature (Tg), upon the removal of the poling electric field. All the decay curves can be fitted by a double exponential function. Below Tg, the slower relaxation time shows an Arrhenius temperature dependence. An extrapolation to room temperature allows to predict the time stability which results to be longer than 30years. In addition, absorption spectra measurements of the films were performed before and immediately after poling procedure to estimate independently the polar order of the dipoles through the decrease of the absorption coefficients.

Photolithography of 3D Scaffolds for Artificial Tissue

The present study is focused on the design and fabrication of novel functional 3D-hydrogel scaffolds for regenerative medicine. In order to critically analyzing the effect of the microarchitecture of 3D scaffolds for driving the cellular fate and diffusion of progenitor stem cells we have fabricate a number of scaffolds with different geometry, stiffness and composition. The physical characteristics of the scaffold determine indeed, as well the biochemical factors, the fate of the cells. We use an innovative composite material consisting of hydrogel with different molecular weight and with suitable accordion-like and woodpile structures in order to tailor stiffness and elasticity conferred to the final structure.These novel 3D bioinspired scaffolds were obtained by both single- (1PP) and two-photon polymerization (2PP) processing. In particular, 2PP scaffolds represent a great advantage with respect to previous achievements based on traditional methods. 3D-structures were fabricated with lateral resolution of some microns, allowing an advanced control of pore microarchitecture of defined tensile strength, and the inclusion of albumin microspheres with various functionalities. The morphological, biochemical and functional characteristics are discussed. Moreover, the effects of the structured hydrogel scaffolds on the proliferation and differentiation of adult stem cells is analyzed in view of the fabrication of portion of contractile cardiac muscle to be obtained In Vitro.

Vapour Sensitivity of InP Surface Quantum Dots

We studied the effect of solvent vapours on the photoluminescent emission of self-assembled InP surface quantum dots (SQDs). Their room temperature near infrared emission undergoes a fully reversible intensity enhancement when the dots were exposed to vapours of polar solvents since polar molecules are likely to be adsorbed onto intrinsic surface states and thus reducing non radiative surface recombination. The shape and position of the emission band does not change. The observed effect is dependent on solvent type and concentration with linear law over a limited concentration range.