Domenico Grasso

Electronic spectra and structure of nitroanilines

Abstract Vapour spectra and solution spectra in a variety of solvents were recorded for some D,D′A r A and A,A′A r D molecules, where DD′NH 2 and AA′NO 2 . Ground state dipole moments of D,D′A r A molecules were determined in dioxane solution. The molecules in molecules (MIM) molecular orbital method was used to calculate the properties associates with the electronic states. It was found that to the first band in the spectra both charge-transfer and locally excited configurations contribute. The dipole moment in the excited state associated with the first intense electronic transition was evaluated from the solvent shift of the band. A general comparison of theoretical results with experiment reveals a good agreement.

Extended theoretical analysis of irreversible protein thermal unfolding

The theoretical analysis of the protein denaturation model which includes an irreversible, exothermic and rate-limited step has been improved and applied to the DSC profile of Azurin. The two-step nature of the irreversible denaturation of globular proteins is usually depicted in the following simplified scheme: N <--> U <--> F, which is known as the Lumry and Eyring model. In most of the works concerning the thermal unfolding of proteins, it is usually assumed that the irreversible step of the process does not take place significantly during the short time the protein spends in the temperature range of the DSC transition, or if this is not the case, that this irreversible step occurs with a negligible thermal effect. As we will show, this last assumption cannot be accepted acritically; in fact we have found that in the case of Azurin an evident exothermic effect occurs at the end of the transition. In order to fit the experimental Cp(exc) profile of Azurin, we have analyzed a model in which the exothermic effects of the irreversible step and the variations of DeltaH with temperature are taken into account. Our model was first tested simulating a series of profiles and considering the effects of the variation of the parameters on the shape of the curves, and successfully used to fit the experimental calorimetric profile of Azurin.

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.

The role of aromatic side-chains in amyloid growth and membrane interaction of the islet amyloid polypeptide fragment LANFLVH

Human islet amyloid polypeptide (hIAPP) is known to misfold and aggregate into amyloid deposits that may be found in pancreatic tissues of patients affected by type 2 diabetes. Recent studies have shown that the highly amyloidogenic peptide LANFLVH, corresponding the N-terminal 12–18 region of IAPP, does not induce membrane damage. Here we assess the role played by the aromatic residue Phe in driving both amyloid formation and membrane interaction of LANFLVH. To this aim, a set of variant heptapeptides in which the aromatic residue Phe has been substituted with a Leu and Ala is studied. Differential scanning calorimetry (DSC) and membrane-leakage experiments demonstrated that Phe substitution noticeably affects the peptide-induced changes in the thermotropic properties of the lipid bilayer but not its membrane damaging potential. Atomic force microscopy (AFM), ThT fluorescence and Congo red birefringence assays evidenced that the Phe residue is not required for fibrillogenesis, but it can influence the self-assembling kinetics. Molecular dynamics simulations have paralleled the outcome of the experimental trials also providing informative details about the structure of the different peptide assemblies. These results support a general theory suggesting that aromatic residues, although capable of affecting the self-assembly kinetics of small peptides and peptide-membrane interactions, are not essential either for amyloid formation or membrane leakage, and indicate that other factors such as β-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.

Calcium-activated membrane interaction of the islet amyloid polypeptide : Implications in the pathogenesis of type II diabetes mellitus

The role played by Ca(2+) ions in the interaction of the human islet amyloid polypeptide (hIAPP) with model membranes has been investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) experiments. In particular, the interaction of hIAPP and its rat isoform (rIAPP) with zwitterionic dipalmitoyl-phosphatidylcholine (DPPC), negatively charged dipalmitoyl-phosphatidylserine (DPPS) vesicles and with a 3:1 mixtures of them, has been studied in the presence of Ca(2+) ions. The experiments have evidenced that amorphous, soluble hIAPP assemblies interact with the hydrophobic core of DPPC bilayers. Conversely, the presence of Ca(2+) ions is necessary to activate a preferential interaction of hIAPP with the hydrophobic core of DPPS membranes. These findings support the hypothesis that an impaired cellular homeostasis of Ca(2+) ions may promote the insertion of hIAPP into the hydrophobic core of carrier vesicles which is thought to contribute to an eventual intracellular accumulation of beta-sheet rich hIAPP aggregates.

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.

Thermodynamics and kinetics of the thermal unfolding of plastocyanin

Abstract The thermal denaturation of plastocyanin in aqueous solution was investigated by means of DSC, ESR and absorbance techniques, with the aim of determining the thermodynamic stability of the protein and of characterizing the thermally induced conformational changes of its active site. The DSC and absorbance experiments indicated an irreversible and kinetically controlled denaturation path. The extrapolation of the heat capacity and optical data at infinite scan rate made it possible to calculate the kinetic and thermodynamic parameters associated with the denaturation steps. The denaturation pathway proposed, and the parameters found from the calorimetric data, were checked by computer simulation using an equation containing the information necessary to describe the denaturation process in detail. ESR and absorbance measurements have shown that structural changes of the copper environment occur during the protein denaturation. In particular, the geometry of the copper-ligand atoms changes from being tetrahedral to square planar and the disruption of the active site precedes the global protein denaturation. The thermodynamic enthalpic change, the half-width transition temperature, and the value of ΔCp, were used to calculate the thermodynamic stability, ΔG, of the reversible process over the entire temperature range of denaturation. The low thermal stability found for plastocyanin, is discussed in connection with structural factors stabilizing the native state of a protein.

Self-Assembling Pathway of HiApp Fibrils within Lipid Bilayers

The presence of insoluble fibrillar deposits in the pancreas is one of the most common pathological features in patients affected by diabetes mellitus type II. The constituent of these deposits is the hormone known as islet amyloid polypeptide (IAPP) or amylin, a 37-amino-acid polypeptide cosecreted and costored with insulin by Langherans b-cells. This hormone plays a central role in the regulation of glucose’s homeostasis. Human amylin (hIAPP) forms amyloid fibrils in vitro; on the other hand, rat amylin (rIAPP), which differs from the human variant by only six amino acid residues, does not form fibrils and is not toxic. The dysfunction and death of Langherans b-cells has been attributed to the conversion from soluble monomers of hIAPP to insoluble b-sheet fibrils, which lead to extracellular buildup. However, recent studies have refuted this hypothesis, and it is unclear whether Langherans cell death is due to the fibrils or to simple structures. In any case, there is experimental evidence, recently reviewed by Jayasinghe and Langen, of the crucial involvement of lipid membranes in the fibrillogenic process. The fibrillogenesis process of hIAPP follows a nucleation-dependent mechanism that comprise a first phase (lag time), in which monomers form small, soluble, unstructured aggregates, 16] followed by a second phase, in which unstructured aggregates transform into b-sheet-rich structures that form the mature fibrils through a self-assembling process. Moreover, it has been suggested that the main mechanism for hIAPP bcell cytotoxicity can be ascribed to membrane permeabilization, through the formation of membrane channels by soluble hIAPP or by hIAPP-induced bilayer disruption (detergent like). In this work, by using hIAPP in the presence of 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes, we provide evidence that the process of membrane destruction is related to the formation of fibrils. POPC lipid membranes were used both as vesicles (LUVs) and supported lipid bilayers (SLBs) in order to simulate the extracellular membrane. Fluorescence spectroscopy, atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) have been employed to characterize the membranes and the interacting hIAPP-membrane systems. In particular, fluorescence versus time measurements have been used to investigate the leakage of a fluorescent probe from LUVs as a test for aspecific membrane pore formations, the thioflavin T (ThT) assay has been used to test the fibril formation, and AFM has been used to obtain morphological information on the studied systems. Finally, the kinetics of hIAPP adsorption onto SLBs have been studied by using QCMD (see the Supporting Information). In Figure 1, the results of fluorescence versus time experiments of hIAPP and 6-carboxyfluorescein-filled LUVs, are shown (red dots). It can be seen that an increase of fluores-

A calorimetric study of the different thermal behaviour of DNA in the isotropic and liquid-crystalline states

Abstract A difference between the thermal behaviour of the isotropic and liquid-crystalline state of sonicated DNA in aqueous salt solution containing poly(ethyleneglycol)(PEG) has been demonstrated. On cooling, a different degree of renaturation of thermally denaturated DNA is observed between samples which form the isotropic state and more concentrated samples which on cooling form the cholesteric state.

The effects of scan rate and protein concentration on DSC thermograms of bovine superoxide dismutase

Abstract DSC thermograms of bovine CuZn superoxide dismutase (BSOD) have been recorded at different scan rates and protein concentrations in order to clarify the process of its unfolding. The lack of calorimetric reversibility made direct thermodynamic analysis of the thermograms impossible. The study of the effect of the scan rate on the shape of the heat capacity (Cp) profiles of BSOD has allowed the calculation of the apparent activation energy (Eapp) for the whole irreversible process. Extrapolation of the excess heat capacity curves (Cpexc) to infinite scan rate has provided the scan-rate-independent part of the thermograms. The protein concentration effect is explained by a change in molecularity that takes place before the kinetically-controlled step. The data collected suggest that, on thermal denaturation, BSOD dissociates during the thermally-induced transitions of the protein; this is followed by a kinetically controlled, exothermic step which is responsible for the global irreversibility of the entire process.