C. La Rosa

A Spectroscopic and Calorimetric Investigation on the Thermal Stability of the Cys3Ala/Cys26Ala Azurin Mutant

The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the proteins overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin.

DSC study of the interaction of the prion peptide PrP106–126 with artificial membranes

The incorporation of the human prion peptide PrP106–126 into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) host model membranes has been investigated by differential scanning calorimetry. Two different types of peptide–membrane interactions have been studied. In one case, the peptide is obliged to be included into the hydrocarbon region of the lipid bilayer, in the other case, it is allowed to interact with the external surface of the membrane. The PrP106–126 incorporated into the DPPC membrane shows an increase in the gel–liquid crystal transition temperature of the bilayer with a decreased enthalpy change. The “oriented” insertion of the hydrophobic part of the fragment into the bilayer, with a consequent increase in the order of the lipidic hydrocarbon chains in the gel state, is responsible for this behavior. Independently from the procedure adopted for the preparation of the sample, the PrP106–126 fragment interacts strongly and irreversibly with the host membrane. In contrast, when PrP106–126 is incorporated in the DPPE model membrane, the gel–liquid crystal transition temperature of the bilayer and the associated enthalpy change decrease. When added externally to the DPPE model membrane, the PrP106–126 fragment has no effect on the phase behavior of the bilayer. These findings suggest that PrP106–126 has a specific affinity for DPPC membranes that might correspond to the external surface of cells.

Experimental model for the thermal denaturation of azurin: a kinetic study

Abstract The thermal denaturation of azurin in H 2 O, D 2 O and in ethanol-H 2 O mixtures has been investigated by electron spin resonance (ESR), optical absorption spectroscopy and differential scanning calorimetry (DSC). The OD 625 T variation observed at a scan rate of 0.7°C/min in H 2 O shows a cooperative OD transition between 78 and 82°C. In this step the intense charge-transfer band of azurin at 625 nm disappears. The ESR spectra recorded at − 153°C of the protein in the native state and after heating at 80 and 82°C indicate that both the symmetry and the copper ligands change with the thermal transition. The DSC measurements show that the thermal denaturation of azurin, which occurs at 84.4°C, is irreversible and kinetically controlled. This complex transition has been described as a multistep denaturation path and was analysed using a Lumry-Eyring type mechanism. The experimental C p.exc profile has been simulated and the calorimetric enthalpies related to the reversible and irreversible step, ΔH u and ΔH ag , respectively, are obtained. The kinetically controlled steps have been investigated by means of optical and DSC measurements at different scan rates and the apparent activation energy, E a , has been calculated. The denaturation of azurin in D 2 O and ethanol-H 2 O mixtures follows the same denaturation path as in H 2 O, although a shift of the OD 625 T and DSC profiles in evidenced. The temperature of the thermal transition and the E a values decrease in ethanol-H 2 O mixtures, but increase in D 2 O.

Phospholipid vesicles as drug delivery systems: Part II. A study on kinetic fusion between vesicles containing CDP-choline and dipalmitoylphosphatidylcholine vesicles

Abstract We have studied the phenomenon of vesicle fusion by differential scanning calorimetric (DSC) and scanning dilatometric (SD) measurements of pure and mixed phospholipids containing CDP-choline. In our study on mixed phospholipid systems, two preparation methods for multilamellar vesicles were used: homogeneous and heterogeneous. The fusion, characteristic of the charged system, is inhibited in the presence of the drug, but occurs when dipalmitoylphosphatidylserine (DPPS) was “diluted” in dipalmitoylphosphatidylcholine (DPPC). In addition, we have evaluated the inclusion efficiency in pure and mixed phospholipids, finding that DPPC/DPPS (3:1 molar ratio) liposomes are the best “hostguest” system, considering the fusion properties and the inclusion efficiency.

Interaction of prion peptide PrP 180-193 with DPPC model membranes: a thermodynamic study

The conversion of Prion (PrPc), a protein normally found on the outer surface of neurons, into an abnormal conformer (PrPsc) is a key molecular event in the pathogenesis of Prion diseases. Recent experimental observations support the hypothesis that an abnormal interaction of PrP with the lipid membrane might be involved in the conversion of PrPc into PrPsc. Particularly, a peptide with an amino acid sequence VNITIKQHTVTTTT corresponding to the second helical region of Prion, PrP 180–193, has been shown to form amyloid aggregates in water and to possess a copper-mediated toxicity to neurons. In the present work, the interaction of PrP 180–193 and its N-terminally acetylated and C-terminally amidated derivative with model membranes formed by dipalmitoylphosphatidylcholine (DPPC) was predicted by thermodynamic models and studied by differential scanning calorimetry (DSC). Thermodynamic predictions give negative values of ΔG only when the peptides transfer, in a structured form, from water to the interface region of the DPPC membrane. The DSC peaks referring to samples prepared according to two different protocols evidence remarkable differences. When the peptides are simply added to an aqueous suspension of the DPPC membrane only the blocked peptide inserts spontaneously; on the other hand when the segments are forced to insert into the membrane, by mixing lipids and peptides during the preparation of the membrane, a different behaviour is evidenced depending on the presence or absence of free charges at the ends of peptides. Our findings suggest a topological situation in which the hydrophobic part of the two peptides inserts into the membrane at a different depth as the copper effect on the complex membrane–peptide system clearly shows. These findings might have intriguing implications on the understanding of the molecular reasons for the neurotoxicity of this prion fragment.

Isothermal compressibility of phospholipid vesicles: A new fast experimental approach

SummaryA simple experimental approach that allows to obtain directly isothermal compressibility values for lyotropic sistems as phospholipid/water is reported. A new sample hold was designed as implementation of a commercial thermomechanical apparatus. Isothermal compressibility values of dipalmitoylphosfatidilcoline/water system and relative volume fluctuations in the different phases are presented.

The effect of copper/zinc replacement on the folding free energy of wild type and Cys3Ala/Cys26Ala azurin

The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation experiments have shown that, in both cases, the thermal transitions of the zinc derivative of azurins can be depicted in terms of the classical Lumry-Eyring model, N if U-->F, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temperature than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temperature. Comparison of the thermodynamic data shows that the presence of Zn in the active site stabilises the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temperature of Zn azurin has increased by 4 degrees C, while the unfolding free energy, deltaG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the observed DeltaG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the reference state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilising effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn derivative, which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, E(a), as derived from the kinetic stability analysis.

A thermodynamic and N.M.R. study of the ribbon lyotropic mesophases. An investigation of the potassium palmitate/potassium laurate/water system

Abstract Lyotropic ternary mixtures of K-palmitate/K-laurate/water with different compositions have been investigated by 2H N.M.R., differential scanning calorimetry and thermo-volumetric analysis. The transitions between rectangular ribbon-like aggregates and lamellar structures have been characterized and explained on the basis of a theoretical model. Our results suggest that for ternary systems containing a small amount of short chain lipids the ribbon structure is more stable at low temperature. Moreover, the rectangular shape is favoured both by the segregation of the short chain lipids at the curved edges of the ribbon and by a tighter packing of the hydrocarbon chains in the same region.

ISOTHERMAL VOLUME VARIATIONS IN LIPID VESICLE SUSPENSIONS - A NEW EVIDENCE OF INTERVESICLE FUSION KINETICS

SummaryIn the present study of fusion between lipid vesicles performed by thermomechanical analysis, isothermal volume variation has been shown to be a reliable tool to follow these kinetics without introducing perturbing probes. In fact, the fusion process is accompanied by bilayer strain release which causes an overall volume decrease of the fused vesicles. Volumetric variations induced by side processes, such as adhesion or ion binding onto the vesicle surface, were accounted for in our measurements. Moreover, by the same technique we followed segregation effects of the membrane lipid components in mixed vesicles. The systems examined were neutral and anionic phospholipids containing vesicles. The role of temperature, vesicle size, lipid composition as well as the influence of different cations were also investigated.

Metal Binding to Prion Protein

The number of original research articles, review articles, book chapters, and monographs published annually on prion is staggering. The experienced reader in this field might ask: “Do we really need another review on prion protein?” This chapter attempts to be different from other recent reviews by focusing on the chemical aspects of metal binding of prion and some of its peptide fragments, rather than the biochemical and biomedical aspects, which have been covered extensively elsewhere [1, 2, 3, 4]. A treatment of the fundamental chemistry should provide a framework for a better understanding of prion function.