Multiple molecular dynamics simulations were performed to investigate the association of stearic ... more Multiple molecular dynamics simulations were performed to investigate the association of stearic acid into the highest affinity binding site of human serum albumin. All binding events ended with a rapid (<10 ps) lock-in of the fatty acid due to formation of a hydrogen bond with Tyr401. The kinetics and energetics of the penetration process both depended linearly on the positional shift of the fatty acid, with an average insertion time and free energy reduction of, respectively, 32 ± 20 ps and 0.70 ± 0.15 kcal/mol per methylene group absorbed. Binding events of longer duration (tbind>1 ns) were characterized by a slow exploration of the pocket entry and, frequently, of a nearby protein crevice corresponding to a metastable state along the route to the binding site. Taken all together, these findings reconstruct the following pathway for the binding process of stearic acid: (i) contact with the protein surface, possibly facilitated by the presence of an intermediate location, (ii) probing of the site entry, (iii) insertion into the protein, and (iv) lock-in at the final position. This general description may also apply to other long-chain fatty acids binding into any of the high-affinity sites of albumin, or to specific sites of other lipid-binding proteins.
Continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy and electron spin echo meth... more Continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy and electron spin echo methods of pulsed EPR of phosphatidylcholine spin-labeled at different positions, n, in the sn-2 chain (n-PCSL, n=5, 7, 10, 12, 14, and 16) are used to study the interaction of inorganic mercury chloride HgCl2 with multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC). For temperatures through the gel phase of DPPC multilayers, the CW-EPR spectra show that an increase of HgCl2 content in the dispersion medium slightly increases the rotational mobility of 5-PCSL and markedly restricts the motion of 16-PCSL. Mercury chloride at 100mM (HgCl2/lipid molar ratio=2:1) removes the gradient of increasing mobility along the chain found in DPPC bilayers in the gel phase. In contrast, HgCl2 does not influence the DPPC chain flexibility profile in the fluid phase. It also suppresses the pre-transition and moderately downshifts the main transition temperature of DPPC membranes. These findings indicate that HgCl2 affects the lipid chain packing of DPPC bilayers and are consistent with the induction of an interdigitated gel phase. Further, D2O-electron spin echo envelope modulation spectroscopy indicates that in the interdigitated phase a higher water permeation is favored at any chain position and the sigmoidal transmembrane water accessibility profile of DPPC bilayers is abolished. Accordingly, the positional dependence of (14)N-hyperfine splitting, 2Azz, shows that the typical hydrophobic barrier of DPPC is significantly altered in the interdigitated phase and all the segments of the lipid chains result to be in a more polar environment.
Thermodynamics of the Thermal Unfolding of Azurin ... Carmelo La Rosa,* Danilo Milardi, and Domen... more Thermodynamics of the Thermal Unfolding of Azurin ... Carmelo La Rosa,* Danilo Milardi, and Domenico Grasso Dipartimento di Scienze Chimiche, Universitb di Catania, V.le A. Doria, 6-95125 Catania, Italy ... Rita Guzzi and Luigi Sportelli Dipartimento di Fisica, Universitb della ...
Abstract. The influence of lysopalmitoylphosphatidylcholine (Lyso-PPC) on lamellar dispersions of... more Abstract. The influence of lysopalmitoylphosphatidylcholine (Lyso-PPC) on lamellar dispersions of dipalmitoylphosphatidyl-choline (DPPC) has been investigated by spectrophotometry and electron spin resonance (ESR) of spin-labelled phosphatidyl-choline at the C-5 or at the ...
For MLVs dispersions, irrespective of the lipid matrix, the presence of Paclitaxel from 1 to 3 mo... more For MLVs dispersions, irrespective of the lipid matrix, the presence of Paclitaxel from 1 to 3 mol% causes the down shift of both pre-(T p ) and main-phase (T m ) transition temperatures and the broadening of the thermograms. The effects are, however, more pronounced on ...
Electron spin-echo envelope modulation (ESEEM) spectroscopy of phospholipids spin-labeled systema... more Electron spin-echo envelope modulation (ESEEM) spectroscopy of phospholipids spin-labeled systematically down the sn-2 chain was used to detect the penetration of water (D2O) into bilayer membranes of dipalmitoyl phosphatidylcholine with and without 50 mol % cholesterol. Three-pulse stimulated echoes allow the resolution of two superimposed 2H-ESEEM spectral components of different widths, for spin labels located in the upper part of the lipid chains. Quantum chemical calculations (DFT) and ESEEM simulations assign the broad spectral component to one or two D2O molecules that are directly hydrogen bonded to the N-O group of the spin label. Classical ESEEM simulations establish that the narrow spectral component arises from nonbonded water (D2O) molecules that are free in the hydrocarbon chain region of the bilayer membrane. The amplitudes of the broad 2H-ESEEM spectral component correlate directly with those of the narrow component for spin labels at different positions down the lipid chain, reflecting the local H-bonding equilibria. The D2O-ESEEM amplitudes decrease with position down the chain toward the bilayer center, displaying a sigmoidal dependence on position that is characteristic of transmembrane polarity profiles established by other less direct spin-labeling methods. The midpoint of the sigmoidal profile is shifted toward the membrane center for membranes without cholesterol, relative to those with cholesterol, and the D2O-ESEEM amplitude in the outer regions of the chain is greater in the presence of cholesterol than in its absence. For both membrane types, the D2O amplitude is almost vanishingly small at the bilayer center. The water-penetration profiles reverse correlate with the lipid-chain packing density, as reflected by 1H-ESEEM intensities from protons of the membrane matrix. An analysis of the H-bonding equilibria provides essential information on the binding of water molecules to H-bond acceptors within the hydrophobic interior of membranes. For membranes containing cholesterol, approximately 40% of the nitroxides in the region adjacent to the lipid headgroups are H bonded to water, of which ca. 15% are doubly H bonded. Corresponding H-bonded populations in membranes without cholesterol are ca. 20%, of which ca. 6% are doubly bonded.
ABSTRACT Conductometric measurements of biological cells in aqueous saline suspensions have been ... more ABSTRACT Conductometric measurements of biological cells in aqueous saline suspensions have been used to investigate the membrane electric parameters which describe the arrangement and the dynamical behaviour of both natural and artificial membranes. Different examples are reported, dealing with the alterations in erythrocyte membrane conductivity following γ-irradiation and with the different transport mechanisms across the erythrocyte membrane in presence of different alkali metal ions. p]Finally, the same technique has been applied to investigate the electrical changes occurring during the fusion of myoblast cells.
International Journal of Biological Macromolecules, 2011
The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of... more The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of Cu(II) and Zn(II) was investigated by using optical absorption, fluorescence, AFM and EPR spectroscopy. Turbidity experiments as a function of temperature indicate that the protein aggregation occurs after the melting of the protein. The kinetic of HSA aggregation, investigated between 60 and 70°C by monitoring the optical density changes at 400nm on a 180min time window, shows an exponential growth with a rate that increases with the temperature. Fluorescence of the thioflavin T evidences a significant increase of the intensity at 480nm at increasing incubation time. These results combined with AFM experiments show that the protein aggregates are elongated oligomers with fibrillar-like features. The absence of a lag-phase suggests that the early stage aggregation of HSA follows a downhill pathway that does not require the formation of an organized nucleus. The presence of Cu(II) and Zn(II) ions does not affect the thermally induced aggregation process and the morphology of HSA aggregates. The result is compatible with the binding of the metal ions to the protein in the native state and with the high conformational stability of HSA.
International Journal of Biological Macromolecules, 2003
The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) an... more 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.
The macrocycles L 1 L 3 having N 2 S 2 O-, N 2 S 2 -, and N 2 S 3 -donor sets, respectively, and... more The macrocycles L 1 L 3 having N 2 S 2 O-, N 2 S 2 -, and N 2 S 3 -donor sets, respectively, and incorporating the 1,10-phenanthroline unit interact in EtOH and MeCN solutions with Cu II to give 1:1 [M(L)] 2+ complex species. The compounds [Cu(L 1 )(ClO 4 )]ClO 4 (1), [Cu(L ...
... References. Benincasa et al., 2003 C. Benincasa, A. De Nino, N. Lombardo, E. Perri, A. Tagare... more ... References. Benincasa et al., 2003 C. Benincasa, A. De Nino, N. Lombardo, E. Perri, A. Tagarelli and G. Sindona, Assay of aroma active components of virgin olive oils from Southern Italian regions by SPME-GC/ion trap mass spectrometry. ...
Site specific spectroscopic techniques and differential scanning calorimetry were used to study h... more Site specific spectroscopic techniques and differential scanning calorimetry were used to study human serum albumin (HSA) in the absence and in the presence of membranes composed of dipalmitoylphosphatidylcholine (DPPC) and poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Electron spin resonance (ESR) of a maleimide spin-label (5-MSL) covalently bound to the free sulfhydryl group at the unique cystein Cys-34 in domain I, intrinsic fluorescence of the single tryptophan Trp-214 in domain II, and extrinsic fluorescence of p-nitrophenyl anthranilate conjugated with tyrosine Tyr-411 in domain III were employed to study HSA dispersions with or without polymer-grafted membranes. On adsorbing at the DPPC membrane surfaces, domain I assumes a more loosened conformation and partitioning of the spin-labelled protein between the aqueous phase and the interfacial region of lipid membranes is observed by ESR. Domain II and III undergo a local structural arrangement which leads Trp-214 and Tyr-411 to come closer and causes intrinsic fluorescence quenching. The influence of DPPC bilayers on HSA is characterized both by a decrease of the thermal unfolding enthalpy and by a slight increase of the transition temperature, Tt, of the protein. The lipid induced effects on HSA are progressively reduced on increasing the amounts of PEG:2000-DPPE mixed with DPPC from the mushroom regime to the brush regime. Primary protein adsorption at the lipid surfaces is abolished at 1 mol% of the polymer-lipid, whereas the secondary protein adsorption at the polymer-brush leads to a further increase of both transition enthalpy and Tt relative to the case of aqueous dispersions of HSA alone.
Abstract The thermal denaturation of plastocyanin in aqueous solution was investigated by means o... more 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 charac-terizing the thermally induced conformational changes of its ...
Two-pulse, echo-detected electron paramagnetic resonance (ED-EPR) spectra and continuous-wave EPR... more Two-pulse, echo-detected electron paramagnetic resonance (ED-EPR) spectra and continuous-wave EPR (CW-EPR) spectra were used to investigate the solvent effect on the librational motion of human haemoglobin spin-labelled on cysteine β93 with the nitroxide derivative of maleimide, 6-MSL. Protein samples fully hydrated in phosphate buffer solution (PBS), in a 60% v/v glycerol/water mixture and in the lyophilized form were measured at cryogenic temperature in the frozen state. The protein librational motion was characterized by the amplitude-correlation time product, <α²>τ(c), deduced from the ED-EPR spectra. The librational amplitude, <α²>τ(c), was determined independently, from the motionally averaged hyperfine splitting in the CW-EPR spectra, and the librational correlation time, τ(c), was derived from the combination of the pulsed and conventional EPR data. Rapid librational motion of small amplitude was detected in all samples. In each case, the librational dynamics was restricted up to 180 K, beyond which it increased steeply for the hydrated protein in PBS and in the presence of glycerol. In contrast, in the dehydrated protein, the librational dynamics was hindered and less dependent on temperature up to ~240 K. In all samples, <α²> deviated from small values only for T > 200 K, where a rapid increase of <α²> was evident for the hydrated samples, whereas limited temperature variation was shown in the lyophilized samples. The librational correlation time was in the sub-nanosecond regime and weakly dependent on temperature. The results evidence that solvent favours protein dynamics.
There is growing evidence that metal ions can accelerate the aggregation process of several prote... more There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of beta-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations </=0.13 mM, the thermogram shows an endothermic peak at 84.3 degrees C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90 degrees C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu(2+) or Zn(2+) ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10 degrees C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein.
Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between... more Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between human serum albumin (HSA) and sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Protein/lipid dispersions are considered in which spin-labelled stearic acids at the 16th carbon atom along the acyl chain (16-SASL) are inserted either in the protein or in the SSL. Two component ESR spectra with different rotational mobility are obtained over a broad range of temperature and membrane composition. Indeed, superimposed to an anisotropic protein-signal, appears a more isotropic lipid-signal. Since in the samples only one matrix (protein or membranes) is spin-labelled, the other component accounts for the transfer of 16-SASL between albumin and membranes. The two components have been resolved and quantified by spectral subtractions, and the fraction, f (p) (16-SASL), of spin labels bound non-covalently to the protein has been used to monitor the transfer. It is found that it depends on the type of donor and acceptor matrix, on the physical state of the membranes and on the grafting density of the polymer-lipids. Indeed, it is favoured from SSL to HSA and the fraction of stearic acids transferred increases with temperature in both directions of transfer. Moreover, in the presence of polymer-lipids, the transfer from HSA to SSL is slightly attenuated, especially in the brush regime of the polymer-chains. Instead, the transfer from SSL to HSA is favoured by the polymer-lipids much more in the mushroom than in the brush regime.
In the present study the thermal unfolding of amicyanin has been addressed using differential sca... more In the present study the thermal unfolding of amicyanin has been addressed using differential scanning calorimetry, fluorescence emission, optical density, circular dichroism and electron paramagnetic resonance. The combined use of these techniques has allowed us to assess, during unfolding of the protein, its global conformational changes in relationship to the local structural modifications occurring in the copper environment and close to the fluorescent chromophore Trp46 of the protein. The thermal transition from the native to the denatured state is on the whole irreversible and occurs in the temperature range between 65 and 72 degrees C, depending on the scan rate and technique used. Amicyanin as a whole shows a complex unfolding pathway, which has been described in terms of a three-step model: N <--> U --> F1 --> F2. According to this model, in the first step the native state of the protein (N) goes reversibly to the unfolded state (U), in the second one U goes irreversibly to F1 and, finally, the state F2 is irreversibly reached in the third step. Kinetic factors prevent the experimental separation of these steps. Nevertheless, the comparison of the data obtained with the different experimental techniques testifies the presence, within the unfolding pathway, of some intermediate states, although not sufficiently long-lived to allow a detailed characterization. A first intermediate transient state has been identified around 68 degrees C, whereas a second one can be related to conformational changes that involve the copper environment. Finally, an exothermal phenomenon, caused by irreversible rearrangements of the melted polypeptide chains, is evidenced. In addition, according to the EPR findings, the type 1 copper ion, which is four-fold coordinated by two N and two S atoms in a distorted tetrahedron in the native state of the protein, shows type 2 features after denaturation. A mathematical model simulating the unfolding Cp(exc) profile has been also developed.
Abstract The thermotropic behavior of dipalmitoylphos-phatidylcholine (DPPC) multibilayers contai... more Abstract The thermotropic behavior of dipalmitoylphos-phatidylcholine (DPPC) multibilayers containing up to 10 mol% of lyso-palmitoylphosphatidylcholine (lyso-PPC) with and without low content of poly(ethylene gly-col:2000)-grafted dipalmitoylphosphatidylethanolamine ...
Multiple molecular dynamics simulations were performed to investigate the association of stearic ... more Multiple molecular dynamics simulations were performed to investigate the association of stearic acid into the highest affinity binding site of human serum albumin. All binding events ended with a rapid (<10 ps) lock-in of the fatty acid due to formation of a hydrogen bond with Tyr401. The kinetics and energetics of the penetration process both depended linearly on the positional shift of the fatty acid, with an average insertion time and free energy reduction of, respectively, 32 ± 20 ps and 0.70 ± 0.15 kcal/mol per methylene group absorbed. Binding events of longer duration (tbind>1 ns) were characterized by a slow exploration of the pocket entry and, frequently, of a nearby protein crevice corresponding to a metastable state along the route to the binding site. Taken all together, these findings reconstruct the following pathway for the binding process of stearic acid: (i) contact with the protein surface, possibly facilitated by the presence of an intermediate location, (ii) probing of the site entry, (iii) insertion into the protein, and (iv) lock-in at the final position. This general description may also apply to other long-chain fatty acids binding into any of the high-affinity sites of albumin, or to specific sites of other lipid-binding proteins.
Continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy and electron spin echo meth... more Continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy and electron spin echo methods of pulsed EPR of phosphatidylcholine spin-labeled at different positions, n, in the sn-2 chain (n-PCSL, n=5, 7, 10, 12, 14, and 16) are used to study the interaction of inorganic mercury chloride HgCl2 with multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC). For temperatures through the gel phase of DPPC multilayers, the CW-EPR spectra show that an increase of HgCl2 content in the dispersion medium slightly increases the rotational mobility of 5-PCSL and markedly restricts the motion of 16-PCSL. Mercury chloride at 100mM (HgCl2/lipid molar ratio=2:1) removes the gradient of increasing mobility along the chain found in DPPC bilayers in the gel phase. In contrast, HgCl2 does not influence the DPPC chain flexibility profile in the fluid phase. It also suppresses the pre-transition and moderately downshifts the main transition temperature of DPPC membranes. These findings indicate that HgCl2 affects the lipid chain packing of DPPC bilayers and are consistent with the induction of an interdigitated gel phase. Further, D2O-electron spin echo envelope modulation spectroscopy indicates that in the interdigitated phase a higher water permeation is favored at any chain position and the sigmoidal transmembrane water accessibility profile of DPPC bilayers is abolished. Accordingly, the positional dependence of (14)N-hyperfine splitting, 2Azz, shows that the typical hydrophobic barrier of DPPC is significantly altered in the interdigitated phase and all the segments of the lipid chains result to be in a more polar environment.
Thermodynamics of the Thermal Unfolding of Azurin ... Carmelo La Rosa,* Danilo Milardi, and Domen... more Thermodynamics of the Thermal Unfolding of Azurin ... Carmelo La Rosa,* Danilo Milardi, and Domenico Grasso Dipartimento di Scienze Chimiche, Universitb di Catania, V.le A. Doria, 6-95125 Catania, Italy ... Rita Guzzi and Luigi Sportelli Dipartimento di Fisica, Universitb della ...
Abstract. The influence of lysopalmitoylphosphatidylcholine (Lyso-PPC) on lamellar dispersions of... more Abstract. The influence of lysopalmitoylphosphatidylcholine (Lyso-PPC) on lamellar dispersions of dipalmitoylphosphatidyl-choline (DPPC) has been investigated by spectrophotometry and electron spin resonance (ESR) of spin-labelled phosphatidyl-choline at the C-5 or at the ...
For MLVs dispersions, irrespective of the lipid matrix, the presence of Paclitaxel from 1 to 3 mo... more For MLVs dispersions, irrespective of the lipid matrix, the presence of Paclitaxel from 1 to 3 mol% causes the down shift of both pre-(T p ) and main-phase (T m ) transition temperatures and the broadening of the thermograms. The effects are, however, more pronounced on ...
Electron spin-echo envelope modulation (ESEEM) spectroscopy of phospholipids spin-labeled systema... more Electron spin-echo envelope modulation (ESEEM) spectroscopy of phospholipids spin-labeled systematically down the sn-2 chain was used to detect the penetration of water (D2O) into bilayer membranes of dipalmitoyl phosphatidylcholine with and without 50 mol % cholesterol. Three-pulse stimulated echoes allow the resolution of two superimposed 2H-ESEEM spectral components of different widths, for spin labels located in the upper part of the lipid chains. Quantum chemical calculations (DFT) and ESEEM simulations assign the broad spectral component to one or two D2O molecules that are directly hydrogen bonded to the N-O group of the spin label. Classical ESEEM simulations establish that the narrow spectral component arises from nonbonded water (D2O) molecules that are free in the hydrocarbon chain region of the bilayer membrane. The amplitudes of the broad 2H-ESEEM spectral component correlate directly with those of the narrow component for spin labels at different positions down the lipid chain, reflecting the local H-bonding equilibria. The D2O-ESEEM amplitudes decrease with position down the chain toward the bilayer center, displaying a sigmoidal dependence on position that is characteristic of transmembrane polarity profiles established by other less direct spin-labeling methods. The midpoint of the sigmoidal profile is shifted toward the membrane center for membranes without cholesterol, relative to those with cholesterol, and the D2O-ESEEM amplitude in the outer regions of the chain is greater in the presence of cholesterol than in its absence. For both membrane types, the D2O amplitude is almost vanishingly small at the bilayer center. The water-penetration profiles reverse correlate with the lipid-chain packing density, as reflected by 1H-ESEEM intensities from protons of the membrane matrix. An analysis of the H-bonding equilibria provides essential information on the binding of water molecules to H-bond acceptors within the hydrophobic interior of membranes. For membranes containing cholesterol, approximately 40% of the nitroxides in the region adjacent to the lipid headgroups are H bonded to water, of which ca. 15% are doubly H bonded. Corresponding H-bonded populations in membranes without cholesterol are ca. 20%, of which ca. 6% are doubly bonded.
ABSTRACT Conductometric measurements of biological cells in aqueous saline suspensions have been ... more ABSTRACT Conductometric measurements of biological cells in aqueous saline suspensions have been used to investigate the membrane electric parameters which describe the arrangement and the dynamical behaviour of both natural and artificial membranes. Different examples are reported, dealing with the alterations in erythrocyte membrane conductivity following γ-irradiation and with the different transport mechanisms across the erythrocyte membrane in presence of different alkali metal ions. p]Finally, the same technique has been applied to investigate the electrical changes occurring during the fusion of myoblast cells.
International Journal of Biological Macromolecules, 2011
The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of... more The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of Cu(II) and Zn(II) was investigated by using optical absorption, fluorescence, AFM and EPR spectroscopy. Turbidity experiments as a function of temperature indicate that the protein aggregation occurs after the melting of the protein. The kinetic of HSA aggregation, investigated between 60 and 70°C by monitoring the optical density changes at 400nm on a 180min time window, shows an exponential growth with a rate that increases with the temperature. Fluorescence of the thioflavin T evidences a significant increase of the intensity at 480nm at increasing incubation time. These results combined with AFM experiments show that the protein aggregates are elongated oligomers with fibrillar-like features. The absence of a lag-phase suggests that the early stage aggregation of HSA follows a downhill pathway that does not require the formation of an organized nucleus. The presence of Cu(II) and Zn(II) ions does not affect the thermally induced aggregation process and the morphology of HSA aggregates. The result is compatible with the binding of the metal ions to the protein in the native state and with the high conformational stability of HSA.
International Journal of Biological Macromolecules, 2003
The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) an... more 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.
The macrocycles L 1 L 3 having N 2 S 2 O-, N 2 S 2 -, and N 2 S 3 -donor sets, respectively, and... more The macrocycles L 1 L 3 having N 2 S 2 O-, N 2 S 2 -, and N 2 S 3 -donor sets, respectively, and incorporating the 1,10-phenanthroline unit interact in EtOH and MeCN solutions with Cu II to give 1:1 [M(L)] 2+ complex species. The compounds [Cu(L 1 )(ClO 4 )]ClO 4 (1), [Cu(L ...
... References. Benincasa et al., 2003 C. Benincasa, A. De Nino, N. Lombardo, E. Perri, A. Tagare... more ... References. Benincasa et al., 2003 C. Benincasa, A. De Nino, N. Lombardo, E. Perri, A. Tagarelli and G. Sindona, Assay of aroma active components of virgin olive oils from Southern Italian regions by SPME-GC/ion trap mass spectrometry. ...
Site specific spectroscopic techniques and differential scanning calorimetry were used to study h... more Site specific spectroscopic techniques and differential scanning calorimetry were used to study human serum albumin (HSA) in the absence and in the presence of membranes composed of dipalmitoylphosphatidylcholine (DPPC) and poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Electron spin resonance (ESR) of a maleimide spin-label (5-MSL) covalently bound to the free sulfhydryl group at the unique cystein Cys-34 in domain I, intrinsic fluorescence of the single tryptophan Trp-214 in domain II, and extrinsic fluorescence of p-nitrophenyl anthranilate conjugated with tyrosine Tyr-411 in domain III were employed to study HSA dispersions with or without polymer-grafted membranes. On adsorbing at the DPPC membrane surfaces, domain I assumes a more loosened conformation and partitioning of the spin-labelled protein between the aqueous phase and the interfacial region of lipid membranes is observed by ESR. Domain II and III undergo a local structural arrangement which leads Trp-214 and Tyr-411 to come closer and causes intrinsic fluorescence quenching. The influence of DPPC bilayers on HSA is characterized both by a decrease of the thermal unfolding enthalpy and by a slight increase of the transition temperature, Tt, of the protein. The lipid induced effects on HSA are progressively reduced on increasing the amounts of PEG:2000-DPPE mixed with DPPC from the mushroom regime to the brush regime. Primary protein adsorption at the lipid surfaces is abolished at 1 mol% of the polymer-lipid, whereas the secondary protein adsorption at the polymer-brush leads to a further increase of both transition enthalpy and Tt relative to the case of aqueous dispersions of HSA alone.
Abstract The thermal denaturation of plastocyanin in aqueous solution was investigated by means o... more 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 charac-terizing the thermally induced conformational changes of its ...
Two-pulse, echo-detected electron paramagnetic resonance (ED-EPR) spectra and continuous-wave EPR... more Two-pulse, echo-detected electron paramagnetic resonance (ED-EPR) spectra and continuous-wave EPR (CW-EPR) spectra were used to investigate the solvent effect on the librational motion of human haemoglobin spin-labelled on cysteine β93 with the nitroxide derivative of maleimide, 6-MSL. Protein samples fully hydrated in phosphate buffer solution (PBS), in a 60% v/v glycerol/water mixture and in the lyophilized form were measured at cryogenic temperature in the frozen state. The protein librational motion was characterized by the amplitude-correlation time product, <α²>τ(c), deduced from the ED-EPR spectra. The librational amplitude, <α²>τ(c), was determined independently, from the motionally averaged hyperfine splitting in the CW-EPR spectra, and the librational correlation time, τ(c), was derived from the combination of the pulsed and conventional EPR data. Rapid librational motion of small amplitude was detected in all samples. In each case, the librational dynamics was restricted up to 180 K, beyond which it increased steeply for the hydrated protein in PBS and in the presence of glycerol. In contrast, in the dehydrated protein, the librational dynamics was hindered and less dependent on temperature up to ~240 K. In all samples, <α²> deviated from small values only for T > 200 K, where a rapid increase of <α²> was evident for the hydrated samples, whereas limited temperature variation was shown in the lyophilized samples. The librational correlation time was in the sub-nanosecond regime and weakly dependent on temperature. The results evidence that solvent favours protein dynamics.
There is growing evidence that metal ions can accelerate the aggregation process of several prote... more There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of beta-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations </=0.13 mM, the thermogram shows an endothermic peak at 84.3 degrees C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90 degrees C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu(2+) or Zn(2+) ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10 degrees C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein.
Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between... more Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between human serum albumin (HSA) and sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Protein/lipid dispersions are considered in which spin-labelled stearic acids at the 16th carbon atom along the acyl chain (16-SASL) are inserted either in the protein or in the SSL. Two component ESR spectra with different rotational mobility are obtained over a broad range of temperature and membrane composition. Indeed, superimposed to an anisotropic protein-signal, appears a more isotropic lipid-signal. Since in the samples only one matrix (protein or membranes) is spin-labelled, the other component accounts for the transfer of 16-SASL between albumin and membranes. The two components have been resolved and quantified by spectral subtractions, and the fraction, f (p) (16-SASL), of spin labels bound non-covalently to the protein has been used to monitor the transfer. It is found that it depends on the type of donor and acceptor matrix, on the physical state of the membranes and on the grafting density of the polymer-lipids. Indeed, it is favoured from SSL to HSA and the fraction of stearic acids transferred increases with temperature in both directions of transfer. Moreover, in the presence of polymer-lipids, the transfer from HSA to SSL is slightly attenuated, especially in the brush regime of the polymer-chains. Instead, the transfer from SSL to HSA is favoured by the polymer-lipids much more in the mushroom than in the brush regime.
In the present study the thermal unfolding of amicyanin has been addressed using differential sca... more In the present study the thermal unfolding of amicyanin has been addressed using differential scanning calorimetry, fluorescence emission, optical density, circular dichroism and electron paramagnetic resonance. The combined use of these techniques has allowed us to assess, during unfolding of the protein, its global conformational changes in relationship to the local structural modifications occurring in the copper environment and close to the fluorescent chromophore Trp46 of the protein. The thermal transition from the native to the denatured state is on the whole irreversible and occurs in the temperature range between 65 and 72 degrees C, depending on the scan rate and technique used. Amicyanin as a whole shows a complex unfolding pathway, which has been described in terms of a three-step model: N <--> U --> F1 --> F2. According to this model, in the first step the native state of the protein (N) goes reversibly to the unfolded state (U), in the second one U goes irreversibly to F1 and, finally, the state F2 is irreversibly reached in the third step. Kinetic factors prevent the experimental separation of these steps. Nevertheless, the comparison of the data obtained with the different experimental techniques testifies the presence, within the unfolding pathway, of some intermediate states, although not sufficiently long-lived to allow a detailed characterization. A first intermediate transient state has been identified around 68 degrees C, whereas a second one can be related to conformational changes that involve the copper environment. Finally, an exothermal phenomenon, caused by irreversible rearrangements of the melted polypeptide chains, is evidenced. In addition, according to the EPR findings, the type 1 copper ion, which is four-fold coordinated by two N and two S atoms in a distorted tetrahedron in the native state of the protein, shows type 2 features after denaturation. A mathematical model simulating the unfolding Cp(exc) profile has been also developed.
Abstract The thermotropic behavior of dipalmitoylphos-phatidylcholine (DPPC) multibilayers contai... more Abstract The thermotropic behavior of dipalmitoylphos-phatidylcholine (DPPC) multibilayers containing up to 10 mol% of lyso-palmitoylphosphatidylcholine (lyso-PPC) with and without low content of poly(ethylene gly-col:2000)-grafted dipalmitoylphosphatidylethanolamine ...
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