The Dinuclear Copper Site Structure of Agaricus bisporus Tyrosinase in Solution Probed by X-ray Absorption Spectroscopy*

We have measured the x-ray absorption near edge structure (XANES) spectra of the enzyme tyrosinase from the mushroom Agaricus bisporus in solution in the oxy and deoxy forms. The spectra, obtained under the same conditions as the analogous forms of mollusc hemocyanin (Hc), show that the oxidation state of copper changes from Cu(II) (oxy form) to Cu(I) (deoxy form), and the copper active site(s) of A. bisporus tyrosinase in solution undergoes the same main conformational changes as Hc. We have applied the multiple scattering theory to simulate the XANES spectra of various alternative geometries of the copper site, accounting for the residual differences between Hc and tyrosinase. While oxy-Hc is reasonably fitted only by the pseudo-square-pyramidal geometry reported by its crystallographic data, oxytyrosinase can be fitted, starting from the Hc coordinates, either by distortions toward a pseudo-tetrahedral geometry, with inequivalent copper sites, or by an apically distorted square-pyramidal geometry (with an elongation of the apical distance of no more than 0.2 A)

Polarized X-ray absorption spectroscopy of the low-temperature photoproduct of carbonmonoxy-myoglobin

Visible light can break the Fe—CO bond in Fe(II) carbonmonoxy-myoglobin (MbCO) giving an unligated product (Mb*) that is almost stable at T < 30 K. Fe K-edge polarized X-ray absorption spectra (P-XAS) of the photoproduct (T = 20 K) of an oriented single crystal (0.2 × 0.2 × 0.3 mm) of sperm whale MbCO (space group P21) have been collected. By rotating the crystal the X-ray photon polarization vector has been oriented almost parallel (with an angle α = 23°) or perpendicular (α = 86°) to the heme normal of each myoglobin molecule. The crystal was continuously illuminated by a white-light source during the data collection. The polarized data give novel information on the Fe-heme electronic/structural rearrangement following photolysis. The XANES (X-ray absorption near-edge structure) spectrum polarized in the direction close to the Fe—CO bond changes dramatically after photolysis, exhibiting a shift of ∼2 eV, due to electronic relaxation of empty states of pz symmetry, while more subtle changes are observed in the spectrum polarized along the heme plane, sensitive to the heme-plane geometry. Changes in the pre-edge region can be interpreted to provide insight into the electronic structure of the highest occupied and lowest unoccupied molecular orbitals (HOMO–LUMO) in the MbCO → Mb* photochemical reaction at low temperature

Glass transition temperature of water confined in lipid membranes as determined by anelastic spectroscopy

The research of gene delivery vehicles used in gene therapy is focused on nonviral vectors like lipid membranes. Such vectors, nonimmunogenic and biodegradable, are formed by complexation of DNA with a mixture of cationic lipids and a neutral colipid which improve the transfection efficiency. A main topic related to lipid membrane dynamics is their capability to spontaneously confine water. At present the value of the glass transition temperature (Tg) is largely debated and determined only by some indirect methods. Here the authors show that anelastic spectroscopy allows the confined water Tg value to be directly identified in several lipid mixtures

Dynamical properties of oriented lipid membranes studied by elastic incoherent neutron scattering

We have studied the H-bonded dynamics in highly oriented lamellar DOTAP-DOPC model membranes as a function of the DOPC/(DOPC + DOTAP) ratio Φ, by elastic incoherent neutron scattering in the 100 ps time domain on the spectrometer IN13 at ILL. Motions in the in-plane and out-of plane directions have been explored by orienting the momentum transfer predominantly parallel and normal to the surface of highly oriented multilayers, respectively. Increasing the neutral lipid (DOPC) concentration from Φ = 0.5-0.8 we reveal a reduction of the dynamics along the direction normal to the membrane and an unchanged dynamics on the membrane plane

Ordering and lyotropic behavior of a silicon-supported cationic and neutral lipid system studied by neutron reflectivity

Self-assembling of amphipathic lipid films on solid support allows the structural investigation of important biological model systems, such as the vectorlike lipid membranes, in order to improve DNA transfection in nonviral gene therapy. We present a neutron reflectivity study of a binary lipid system composed of dioleoylphosphatidylcholine (DOPC) and dimethyldioctadecylammonium bromide (DDAB) deposited on [100] silicon support by means of spin coating technique. We underline their lyotropic behavior under saturated deuterium oxide (D2 O) vapor thus pointing out that the lipid mixture is organized in ordered domains composed of plane lamellar bilayers of noninteractive DOPC and DDAB. © 2008 American Institute of Physics

Correlation between Structure and Transfection Efficiency: a Study of DC-Chol-DOPE/DNA Complexes

The supramolecular structural nature of some cationic liposomes–DNA complexes, currently used as vehicles in non-viral gene delivery, has been elucidated by recent X-ray diffraction experiments. The relationship between the chemico-physical properties of these self-assembled structures and their transfection efficiency is extensively studied. Here we report a first comprehensive structural study by using energy dispersive X-ray diffraction, of the complex DC-Chol−DOPE/DNA (3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol dioleoylphosphatidylethanolamine/DNA), which has been classified as one of the most effective in in-vivo experiments. Our results show that DC-Chol−DOPE/DNA lipoplexes have a columnar inverted hexagonal structure, which is not influenced by the cationic liposome/DNA charge ratio. The transfection efficiency of C6 rat glioma cells by DC-Chol–DOPE/DNA lipoplexes and the toxicity of lipoplexes to cells are dramatically affected by cationic liposome/DNA weight ratio.It seems therefore that the lipoplex structures have not any influence on transfection efficiency and toxicity in our experimental system

UV-B radiation induced effects on human T-lymphocytes

The reactive oxygen species (ROS) produced inside cells by UV-B radiation may induce apoptosis, a process that realizes the programmed death of cells. In the present paper the UV induced damage was studied in a human T-Lymphocyte cell line (the Jurkat line) by mean of IR laser photoacoustic spectroscopy (PAS) and by Fourier Transform Infrared Spectroscopy (FTIR) combined with biological assays based on flow cytometry. The apoptosis was induced in vitro in the Jurkat samples by exposition to UV-B radiation with a dose of 310 mJ/cm2. PAS measurements were performed through a 10W c.w. CO2 laser based optical system realised at ENEA Molecular Spectroscopy Laboratory in Frascati (Italy)

Particle Size Distribution in DMPC Vesicles Solutions Undergoing Different Sonication Times

Size distribution of dimyristoylphosphatidylcholine liposome suspensions was investigated by dynamic-light scattering (DLS) as a function of the sonication time (t(s)). Cumulant expansion (second- and third-order) and regularized Laplace inversion (CONTIN) of dynamic single-angle laser light-scattering data were performed. With both methods, the intensity-weighted mean hydrodynamic radius 〈r〉(I) depended on the investigated lengthscale. The number-weighted mean hydrodynamic radius (〈r〉(N)), obtained from CONTIN by modeling dimyristoylphosphatidylcholine vesicles as thin-walled hollow spheres, resulted as independent on the lengthscale. However, the 〈r〉(N) value obtained from cumulant expansions remained lengthscale-dependent. Therefore, the number-weighted radius distribution function is highly asymmetric. The number-weighted mean radius, the standard deviation, and the number-weighted radius at the peak [Formula: see text] all decreased to a plateau when increasing sonication time. At t(s) longer than 1 h, the [Formula: see text] compares well with the radius of unilamellar vesicles in equilibrium with monomers predicted on a thermodynamic basis. The reliability of our analysis is proved by the comparison of experimental Rayleigh ratios with simulated ones, using the normalized number-weighted radius distribution function p(N)(r) determined by DLS data. A perfect agreement was obtained at longer sonication times, and the average aggregation number was determined. At lower t(s) values, simulations did not match experimental data, and this discrepancy was ascribed to the presence of large and floppy unilamellar vesicles with ellipsoidal shapes. Our investigation shows that, from single-angle DLS data, the radius distribution function of the vesicles can only be obtained if p(N)(r) is known