F. Boffi

Self-assembly of cationic liposomes–DNA complexes: a structural and thermodynamic study by EDXD

Abstract We report a comprehensive study of the lamellar dioleoyl trimethylammonium propane and DNA complexes (DOTAP–DNA) as a function of the lipid/DNA weight ratio ρ in order to determine their structure and thermodynamic stability using an energy dispersive X-ray diffraction technique (EDXD). These self-assemblies consist of a periodic multilayer structure with DNA chains adsorbed between cationic membranes. Furthermore, above and below the isoelectric point, the DNA packing density significantly varies as a function of ρ . Our results confirm that EDXD is a powerful technique to characterize self-assembled ordered structures of oppositely charged macromolecules.

pH-Dependent Local Structure of Ferricytochrome c Studied by X-Ray Absorption Spectroscopy

We have studied, using x-ray absorption spectroscopy by synchrotron radiation, the native state of the horse heart cytochrome c (N), the HCl denatured state (U(1) at pH 2), the NaOH denatured state (U(2) at pH 12), the intermediate HCl induced state (A(1) at pH 0.5), and the intermediate NaCl induced state (A(2) at pH 2). Although many results concerning the native and denatured states of this protein have been published, a site-specific structure analysis of the denatured and intermediate solvent induced states has never been attempted before. Model systems and myoglobin in different states of coordination are compared with cytochrome c spectra to have insight into the protein site structure in our experimental conditions. New features are evidenced by our results: 1) x-ray absorption near edge structure (XANES) of the HCl intermediate state (A(1)) presents typical structures of a pentacoordinate Fe(III) system, and 2) local site structures of the two intermediate states (A(1) and A(2)) are different.

Aluminum site structure in serum transferrin and lactoferrin revealed by synchrotron radiation X-ray spectroscopy

The Al site structure of serum transferrin and lactoferrin is investigated using X-ray absorption near edge structure (XANES) spectroscopy. Al K-edge spectra in the mono- and dialuminum forms of the proteins have been recorded for the first time. Our results show that the aluminium ion is hexa-coordinated in an octahedral-like symmetry and that the monoaluminum form, where only the C-terminal binding site is saturated, has an increased structural distortion around the metal site.

Light induced states in MbCO denatured with guanidine hydrochloride.

Abstract We present the results of a comparative study of the binding of carbon monoxide to myoglobin in glycerol/buffer solution with different concentrations of guanidine hydrochloride, under extended illumination over the temperature range 30 – 80 K. The changes in the Soret band indicate that the folding state of the protein is a key parameter in determining the photodissociation process and the relaxation rate of the protein.

A STRUCTURAL AND KINETIC STUDY BY ENERGY DISPERSION X-RAY DIFFRACTION : INTERACTION BETWEEN 1,4-DIHYDROPYRIDINES AND BIOLOGICAL MEMBRANES

Abstract Application of energy dispersive X-ray diffraction to the study of the main transition phase of the pure and doped DMPC ( l α -dimyristoylphosphatidylcholine; 1,2-ditetradecanoyl- sn -glycero-3-phosphatidylcholine) bilayers is reported. The aim is to provide an investigative tool to follow the structural variation induced in these systems by temperature and by insertion of doping substances. Hydrophobic compounds such as 1,4-dihydropyridines are used as doping molecules; Nifedipine and Lacidipine show different physical–chemical properties, modifying in a different way the membrane bilayer and its main transition phase. The results show that this technique is useful for studying these kinds of systems (membrane bilayer or biopolymer), giving information on structural variations that occur in a relatively short time.

Iron and copper K-edge XAS study of serotransferrin and ovotransferrin

The active metal site structure of transferrin with iron and copper atoms is investigated using metal K-XANES. Theoretical analysis of experimental data has been performed on the basis of full multiple-scattering theory. This approach made it possible to study the origin of XANES fine details and to investigate the local structure around active metal sites. A deep insight into the local structure and electronic subsystem of Fe, Cu transferrins is obtained. For example, in the case of Cu substitution of Fe in the active centre, the best fit of theoretical spectra to experiment has been obtained for distances 3% smaller between the Cu atom and the nearest neighbours.

Comparative XANES study of serotransferrin and ovotransferrin at Cu K-edge: evidence of interactions among the metal sites.

The Cu site structure of human serotransferrin and hen ovotransferrin using XANES spectroscopy has been investigated. Although the transferrin family proteins have been extensively studied, the results reported herein are the first concerning the structure of the metal site in C-terminal and N-terminal in the whole protein. Our structural data show that these proteins differ with regard to the independence of the two binding sites and the geometry of copper coordination, ranging from a poorly to a significantly distorted octahedron.

Structural differences of ovalbumin and S-ovalbumin revealed by denaturing conditions.

We found, by circular dichroism and Raman spectroscopy measurements, that the secondary structure of the native ovalbumin and of its heat-stable form, called S-ovalbumin, is a probe of the structural differences between the two proteins. Small angle X-ray scattering and circular dichroism measurements performed on the two proteins under denaturing conditions, with different concentrations of guanidine hydrochloride, show the changes of the tertiary and secondary structure and a different pathway in the unfolding process. These experimental data confirm that the conversion of native ovalbumin into S-ovalbumin is irreversible and reveal that the response of the two proteins to the same chemical environment is different