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.

Fatty acid binding into the highest affinity site of human serum albumin observed in molecular dynamics simulation.

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.

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.

Librational fluctuations in protein glasses.

Librational motions in the region of the protein "glass" (or dynamic) transition are analysed for spin-labelled haemoglobin, serum albumin and β-lactoglobulin by EPR spectroscopy. A discontinuity in the temperature dependence of the mean-square librational amplitude, <α(2)>, occurs in the region of 200K as found for the mean-square atomic displacement, , at the protein dynamic transition by Mössbauer spectroscopy and neutron scattering. The discontinuity in <α(2)> vs.

Influence of stearic acids on resveratrol-HSA interaction.

The interaction between the natural polyphenol resveratrol and human serum albumin (HSA), the most abundant transport protein in plasma, has been studied in the absence and in the presence of up to six molecules of stearic acids (SA) pre-complexed with the protein. The study has been carried out by using the intrinsic fluorescence of both HSA and resveratrol. Protein and polyphenol fluorescence data indicate that resveratrol binds to HSA with an association constant k(a) = (1.

The influence of active site loop mutations on the thermal stability of azurin from Pseudomonas aeruginosa.

The copper site and overall structures of azurin (AZ) variants in which the amicyanin (AMI) and plastocyanin (PC) metal binding loops have been introduced, AZAMI and AZPC, respectively, are similar to that of AZ, whereas the loop conformations resemble those in the native proteins. To assess the influence of these loop mutations on stability, the thermal unfolding of AZAMI and AZPC has been investigated by differential scanning calorimetry, absorption and fluorescence spectroscopy. The calorimetric profiles of both variants exhibit a complex shape consisting of two endothermic peaks and an exothermic peak.

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.

Solvent effect on librational dynamics of spin-labelled haemoglobin by ED- and CW-EPR.

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.

Spin-echo EPR of Na,K-ATPase unfolding by urea.

Denaturant-perturbation and pulsed EPR spectroscopy are combined to probe the folding of the membrane-bound Na,K-ATPase active transport system. The Na,K-ATPase enzymes from shark salt gland and pig kidney are covalently spin labelled on cysteine residues that either do not perturb or are essential to hydrolytic activity (Class I and Class II -SH groups, respectively). Urea increases the accessibility of water to the spin-labelled groups and increases their mutual separations, as recorded by D2O interactions from ESEEM spectroscopy and instantaneous spin diffusion from echo-detected EPR spectra, respectively.

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.

Molecular dynamics of amicyanin reveals a conserved dynamical core for blue copper proteins.

Molecular dynamics simulation has been carried out for the blue copper protein amicyanin from two different sources, Paracoccus denitrificans and Paraccocus versutus, to investigate the structural and dynamical properties common to the two molecules and to identify prominent features shared with proteins of the same family, the monomeric cupredoxins. The two amicyanins have almost identical secondary and tertiary structure. In the simulation, they differ for the number of hydrogen bonds in the main chain and the conformation of some beta-strands.

Thermal unfolding studies of a phytocyanin.

The thermal stability of umecyanin, a stellacyanin from horseradish roots, has been investigated by differential scanning calorimetry, optical absorption and fluorescence spectroscopy at neutral and alkaline pH. Above pH 9 the Cu(II) protein experiences a blue shift of the main visible absorption band at approximately 600 nm and changes colour from blue to violet. The thermal transition of the protein is irreversible and occurs between 61.

Spectroscopic and calorimetric studies on the interaction of human serum albumin with DPPC/PEG:2000-DPPE membranes.

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.

Backbone dynamics of alamethicin bound to lipid membranes: spin-echo electron paramagnetic resonance of TOAC-spin labels.

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes.

The role played by the alpha-helix in the unfolding pathway and stability of azurin: switching between hierarchic and nonhierarchic folding.

The role played by the alpha-helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying a helix-depleted azurin variant produced by site-directed mutagenesis. The protein structure was investigated by CD, 1D (1)H NMR, fluorescence spectroscopy measurements and MD simulations, whilst EPR, UV-visible and cyclic voltammetry experiments were carried out to investigate the geometry and the properties of the Cu(II) site. The effects of the alpha-helix depletion on the thermal stability and the unfolding pathway of the protein were determined by DSC, UV/visible and fluorescence measurements at increasing temperature.

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.

Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin.

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D(2)O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature.

Structural, dynamical and functional aspects of the inner motions in the blue copper protein azurin.

Molecular dynamics was applied to dissect out the internal motions of azurin, a copper protein performing electron transfer. Simulations of 16.5 ns were analyzed in search of coordinated displacements of amino acid residues that are important for the protein function.

Pro-oxidant activity of oleuropein determined in vitro by electron spin resonance spin-trapping methodology.

In this work, the pro-oxidant behavior of oleuropein (OLP, 1) is characterized in a Fenton-like experiment by means of ESR spectroscopy using the spin trap system DMSO and 4-(pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) in phosphate buffer (PB) solution. Ferrous ions in the absence of hydrogen peroxide cause the formation of the stable nitroxide species 4 and 5 through the intermediate perferryl species. OLP displays its antioxidant activity in vitro blocking the oxidation path that leads to methoxyl radicals hence to the formation of the stable radical species 5.

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.

Water concentration profiles in membranes measured by ESEEM of spin-labeled lipids.

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.

Time-resolved electron spin resonance studies of spin-labelled lipids in membranes.

Recently, developments in time-resolved spin-label electron spin resonance (ESR) spectroscopy have contributed considerably to the study of biomembranes. Two different applications of electron spin echo spectroscopy of spin-labelled phospholipids are reviewed here: (1) the use of partially relaxed echo-detected ESR spectra to study the librational lipid-chain motions in the low-temperature phases of phospholipid bilayers; (2) the use of electron spin echo envelope modulation spectroscopy to determine the penetration of water into phospholipid membranes. Results are described for phosphatidylcholine bilayer membranes, with and without equimolar cholesterol, that are obtained with phosphatidylcholine spin probes site-specifically labelled throughout the sn-2 chain.

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.

Transfer of stearic acids from albumin to polymer-grafted lipid containing membranes probed by spin-label electron spin resonance.

Human serum albumin (HSA) has been spin-labelled with stearic acids having the nitroxide moiety attached to the hydrocarbon chain either at the 5th or at the 16th carbon atom (n-SASL, n = 5 and 16, respectively) with respect to the carboxyl groups. Its interaction with sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) mixed with submicellar content of poly(ethylene glycol:2000)-grafted dipalmitoyl phosphatidylethanolamine (PEG:2000-DPPE) has been studied by conventional electron spin resonance (ESR) spectroscopy. In the absence of bilayer membranes, the ESR spectra of nitroxide stearic acids non-covalently bound to HSA are single component powder patterns, indicative of spin labels undergoing temperature dependent anisotropic motion in the slow motional regime on the conventional ESR timescale.

The early steps in the unfolding of azurin.

High-temperature molecular dynamics simulations were used to gain insight into the early steps in the unfolding pathway of azurin, a blue copper protein with a beta-barrel structure formed by two sheets arranged in a Greek key folding topology. The results reveal that unfolding of the beta-barrel in azurin is associated with dislocation of its unique alpha-helix with respect to the protein scaffold. Exposure of the hydrophobic core to solvent precedes complete disruption of secondary and tertiary structure, and modifications in the region around the active site are directly connected with this event.