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

Structural characterization of a new lipid/DNA complex showing a selective transfection efficiency in ovarian cancer cells

We investigated, for the first time, by using Energy Dispersive X-ray Diffraction, the structure of a new ternary cationic liposome formulated with dioleoyl trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE) and cholesterol (Chol) (DDC) which has been recently found to have a selective high gene transfer ability in ovarian cancer cells. Our structural results provide a further experimental support to the widely accepted statement that there is not a simple and direct correlation between structure and transfection efficiency and that the factors controlling cationic lipid/DNA (CL-DNA) complexes-mediated gene transfer depend not only on the formulations of the cationic liposomes and their thermodynamic phase, but also significantly on the cell properties. Document Type: Articl

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

XAS characterization of the Zn site of non-structural protein 3 (NS3) from hepatitis C virus

XANES spectra of non structural protein 3 (NS3) have been calculated using 4 Zn coordination models from three crystallographic structures in the Protein Data Base (PDB): 1DY9, subunit B, 1CU1 subunit A and B, and 1JXP subunit B. Results indicate that XANES is an appropriate tool to distinguish among them. Experimental XANES spectra have been simulated refining crystallographic data. The model obtained by XAS is compared with the PDB models. © 2007 American Institute of Physics

A new approach for the study of cationic lipid-DNA complexes by EDXD

X-ray diffraction (XRD) studies on the cationic liposomes (CL)-DNA complexes currently used in gene delivery have provided detailed structural informations on these compact, ordered self-assemblies sheding light on the poorly understood structure-activity relationship. Among these, the experiments carried out by using a synchrotron radiation source have showed an experimental resolution remarkably better than that achieved one by traditional in house apparatuses. Here we show a new experimental approach for the study of CL-DNA complexes, based on the employment of silicon wafers as substrates, which allows to obtain high-resolution structural informations by energy dispersive X-ray diffraction (EDXD)

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