Resveratrol induces thermal stabilization of human serum albumin and modulates the early aggregation stage.

Several phenolic compounds bind to proteins and show the ability to interfere with their aggregation process. The impact of the natural polyphenol resveratrol on the stability and heat induced aggregation of human serum albumin (HSA) was investigated by differential scanning calorimetry (DSC), attenuated total reflectance Fourier transform infrared (ATR-FTIR), UV-vis absorbance, ThT fluorescence, atomic force microscopy (AFM) and molecular modeling. The binding of resveratrol to HSA improves the stability of the protein to thermal unfolding, particularly for the energetic domain containing the ligand binding site, as modeled by computational techniques. The thermal unfolding is irreversible and after the melting the protein aggregates, either with or without the ligand. The kinetics of HSA aggregation between 70 and 80°C shows an exponential growth of the absorbance change and it slows down when resveratrol is added. The aggregates have fibril-like morphology and resveratrol attenuates the formation of ß-structured species. The overall results suggest that resveratrol stabilizes the protein structure and modulates the formation of fibrils along the initial stage of the HSA aggregation pathway.

Chain interdigitation in DPPC bilayers induced by HgCl2: evidences from continuous wave and pulsed EPR.

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.

Early stage aggregation of human serum albumin in the presence of metal ions.

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.

Spontaneous transfer of stearic acids between human serum albumin and PEG:2000-grafted DPPC membranes.

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.

Thermal stability effects of removing the type-2 copper ligand His306 at the interface of nitrite reductase subunits.

Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate, N(3)<--(k)-->U(3)--(k)-->F (N-native, U-unfolded and F-final). To understand the role of interfacial residues on protein stability, a type-2 copper site ligand, His306, has been mutated to an alanine. The characterization of the native state of the mutated protein highlights that this mutation prevents copper ions from binding to the type-2 site and eliminates catalytic activity. No significant alteration of the geometry of the type-1 site is observed. Study of the thermal denaturation of this His306Ala NiR variant by differential scanning calorimetry shows an endothermic irreversible profile, with maximum heat absorption at T (max) approximately equal to 85 degrees C, i.e., 15 degrees C lower than the corresponding value found for wild-type protein. The reduction of the protein thermal stability induced by the His306Ala replacement was also shown by optical spectroscopy. The denaturation pathway of the variant is compatible with the kinetic model N(3)--(k)-->F(3), where the protein irreversibly passes from the native to the final state. No evidence of subunits' dissociation has been found within the unfolding process. The results show that the type-2 copper sites, situated at the interface of two monomers, significantly contribute to both the stability and the denaturation mechanism of NiR.

A comparative investigation of the thermal unfolding of pseudoazurin in the Cu(II)-holo and apo form.

The contribution of the copper ion to the stability and to the unfolding pathway of pseudoazurin was investigated by a comparative analysis of the thermal unfolding of the Cu(II)-holo and apo form of the protein. The unfolding has been followed by calorimetry, fluorescence, optical density, and electron paramagnetic resonance (EPR) spectroscopy. The thermal transition of Cu(II)-holo pseudoazurin is irreversible and occurs between 60.0 and 67.3 degrees C, depending on the scan rate and technique used. The denaturation pathway of Cu(II)-holo pseudoazurin can be described by the Lumry-Eyring model: N --> U --> [corrected] F; the protein reversibly goes from the native (N) to the unfolded (U) state, and then irreversibly to the final (F) state. The simulation of the experimental calorimetric profiles, according to this model, allowed us to determine the thermodynamic and kinetic parameters of the two steps. The DeltaG value calculated for the Cu(II)-holo pseudoazurin is 39.2 kJ.mol(-1) at 25 degrees C. The sequence of events in the denaturation process of Cu(II)-holo pseudoazurin emergence starts with the disruption of the copper site and the hydrophobic core destabilization followed by the global protein unfolding. According to the EPR findings, the native type-1 copper ion shows type-2 copper features after the denaturation. The removal of the copper ion (apo form) significantly reduces the stability of the protein as evidenced by a DeltaG value of 16.5 kJ.mol(-1) at 25 degrees C. Moreover, the apo Paz unfolding occurs at 41.8 degrees C and is compatible with a two-state reversible process N --> [corrected] U.

Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase.

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T(m) approximately 100 degrees C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry-Eyring model, N<-->U-->F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed.