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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 100 mM (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 14N-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.

The interaction of saturated fatty acids of different length (C8:0 to C18:0) with β-lactoglobulin (βLG) was investigated by molecular dynamics simulation and docking approaches. The results show that the presence of such ligands in the hydrophobic central cavity of βLG, known as the protein calyx, determines an enhancement of atomic fluctuations compared with the unliganded form, especially for loops at the entrance of the binding site. Concerted motions are evidenced for protein regions that could favor the binding of ligands. The mechanism of anchoring of fatty acids of different length is similar for the carboxylate head-group, through electrostatic interactions with the side chains of Lys60/Lys69. The key protein residues to secure the hydrocarbon chain are Phe105/Met107, which adapt their conformation upon ligand binding. In particular, Phe105 provides an additional hydrophobic clamp only for the tail of the two fatty acids with the longest chains, palmitic, and stearic acid, which are known to bind βLG with a high affinity. The search of additional external binding sites for fatty acids, distinct from the calyx, was also carried out for palmitic acid. Two external sites with a lower affinity were identified as secondary sites, one consisting in a hydrophobic cavity allowing two distinct binding modes for the fatty acid, and the other corresponding to a surface crevice close to the protein α-helix. The overall results provide a comprehensive picture of the dynamical behavior of βLG in complex with fatty acids, and elucidate the structural basis of the binding of these physiological ligands.

Two molecular dynamics simulations have been performed for 2 ns, at room temperature, on fully hydrated wild type and Cys3Ala/Cys26Ala double-mutant azurin, to investigate the role of the unique disulfide bridge on the structure and dynamics of the protein. The results show that the removal of the [bond]SS[bond] bond does not affect the structural features of the protein, whereas alterations of the dynamical properties are observed. The root mean square fluctuations of the atomic positions are, on average, considerably reduced in the azurin mutant with respect to the wild type form. The number of intramolecular hydrogen bonds between protein backbone atoms that are lost during the simulation, with respect to the starting configuration, are reduced in the absence of the disulfide bond. The analysis of the dynamical cross-correlation map, characterising the protein co-ordinated internal motions, demonstrates in the mutated azurin a significant decrease in anti-correlated displacements between protein residues, with the only exception occurring in the region of the mutation sites. The overall findings show a relevant reduction in flexibility as a consequence of the disulfide bridge depletion in azurin, suggesting that the [bond]SS[bond] bond is a structural element which significantly contributes to the dynamic properties of the native protein.

The effect of heavy water on the structure and dynamics of copper plastocyanin as well as on some aspects of the solvent dynamics at the protein–solvent interfacial region have been investigated by molecular dynamics simulation. The simulated system has been analyzed in terms of the atomic root mean square deviation and fluctuations, intraprotein H-bond pattern, dynamical cross-correlation map and the results have been compared with those previously obtained for plastocyanin in H 2 O (Ciocchetti et al. Biophys. Chem. 69 (1997), 185–198). The simulated plastocyanin structure in the two solvents, averaging 1 ns, is very similar along the β-structure regions, while the most significant differences are registered, analogous to the turns and the regions likely involved in the electron transfer pathway. Moreover, plastocyanin in D 2 O shows an increase in the number of both the intraprotein H-bonds and the residues involved in correlated motions. An analysis of the protein–solvent coupling evidenced that D 2 O makes the H-bond formation more difficult with the solvent molecules for positively charged and polar residues, while an opposite trend is observed for negatively charged residues. On the other hand, the frequency of exchange of the solvent molecules involved in the protein–solvent H-bond formation is significantly depressed in D 2 O. The results are discussed also in connection with protein functionality and briefly with some experimental results connected with the thermostability of proteins in D 2 O.

The effects of cooling rate and of solvent properties on the active site heterogeneity of two copper proteins, azurin and plastocyanin, have been investigated at low temperature by electron paramagnetic resonance spectroscopy. The spectra of theses proteins have been analyzed, by an accurate computer simulation, in terms of a distribution of some relevant spin-Hamiltonian parameters. The results show that the structural heterogeneity of both proteins, quantified by the width of the distribution in the g and A tensors, is affected by both the freezing procedure and the solvent composition. In particular, the g distribution width is found to be reduced in the slow cooling regime; such a reduction appearing more significant when glycerol is added to the protein solutions. Despite of the similarity in the copper ion microenvironments of the two proteins, the effects are more pronounced in azurin. The results are discussed also in connection with the role played by the solvent and the rate of freezing in featuring the conformational substate landscape.

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.

Librational motions in the region of the protein "glass" (or dynamic) transition are analysed for spin-labelled haemoglobin, serum albumin and beta-lactoglobulin by EPR spectroscopy. A discontinuity in the temperature dependence of the mean-square librational amplitude, , occurs in the region of 200 K as found for the mean-square atomic displacement, , at the protein dynamic transition by Mossbauer spectroscopy and neutron scattering. The discontinuity in vs. T can be described by the Vogel-Tammann-Fulcher equation, implying a finite glass transition temperature. Above the dynamic transition, vs. 1/T can be approximated by the Arrhenius law with activation energies similar to those usually found for , and relaxation processes in glass-forming media and the hydration shells of proteins. Similar results are found for librational fluctuations of membranous Na,K-ATPase spin-labelled either on superficial -SH groups or on those essential to activity.

We have investigated by means of electron spin resonance (ESR) spectroscopy using two spin labels, Iodoacetamido-proxyl and 3-Maleimido-proxyl, the dynamics of two different regious around the active site of azurin, a copper containing blue protein. The ESR measurements of spin labeled azurin have been carried out in the 110–300 K temperature range on wet (H2O, D2O and ethanol/water mixtures) and lyophilized samples. The behaviours of the outer hyperfine splitting separation, 2A′ zz , of the ESR spectravs temperature of the lyophilized, and fully hydrated azurin in H2O and D2O suggest that the two spin labels are located in regions of the protein surface with different dynamics and polarity. Moreover, all differences in the 2A′ zz values shown by the spin labeled azurin in normal and heavy water as well as the temperature behaviour disappear when azurin is in ethanol/water mixtures. The results are discussed in terms of a close correlation between the molecular dynamics of the protein fragments to which the two spin labels are bound and the properties of the solvent used.

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. The greater effects of urea are experienced by Class I groups, which indicates preferential unfolding of the extramembrane domains. Conformational heterogeneity induced by urea causes dispersion in spin-echo phase-memory times to persist to higher temperatures. Analysis of lineshapes from partially relaxed echo-detected EPR spectra indicates that perturbation by urea enhances the amplitude and rate of fluctuations between conformational substates, in the higher temperature regime, and also depresses the glasslike transition in the protein. These non-native substates that are promoted by urea lie off the enzymatic pathway and contribute to the loss of function.

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 400 nm on a 180 min 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 480 nm 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.

Human serum albumin provides the transport of long-chain fatty acids in the blood through three high-affinity and four low-affinity binding sites. Molecular dynamics simulations have been performed to investigate the anchoring of palmitic acid molecules to the protein. In the site with the highest affinity, Site 5, the key residue Lys525 not only binds the head-group of the palmitate ion by electrostatic interactions with its charged terminal group, but it also accommodates the first portion of the lipid chain by non-electrostatic interactions with the rest of its sidechain. The flexibility of Lys525, and in particular of the dihedral angleχ3, is suggested to account for a number of spectroscopic properties observed in correspondence with the entrance of the hydrophobic pocket constituting Site 5.

The interaction of Azurin, a metal-protein with oxidase activity containing one Cu++ ion of type I, with short-chain n- and iso-alcohols has been investigated by electron spin resonance (ESR), optical absorption, and fluorescence emission spectroscopies. The ESR spectra of the protein recorded at 85 K do not show significant variation of the shape of the Cu++ resonance signal even at the highest molar fraction of alcohols present in solution. The optical density of the charge transfer transition of azurin at λ = 625 nm and the fluorescence emission at λ = 308 nm of the unique tryptophan residue present in the protein decrease in the presence of increasing alcohols' molar fraction. The minima values reached depend on the alcohols' properties. They occur, in fact, at the molar fraction value of about 0.01 in the presence of propanol and butanol in the n- and iso- forms and at the value of about 0.03 in the presence of methanol and ethanol. Moreover, the intensity of the CT-band and the fluorescence emission is completely recovered in the presence of the highest alcohol concentrations used. The experimental findings suggest that the Cu++-N2SS* active site of the enzyme is not directly involved in the interaction with the alcohols. The short-chain alcohols at the lower molar fractions change the hydrogen bonds network at the protein-bulk solution interface stabilizing, therefore, a particular conformation (or substate) of the enzyme while, at the highest ones the effects observed are connected with the properties of the binary alcohol-water mixture.

Alamethicin is a 20-residue, hydrophobic, helical peptide, which forms voltage-sensitive ion channels in lipid membranes. The helicogenic, nitroxyl amino acid TOAC was substituted isosterically for Aib at residue positions 1, 8, or 16 in a F50/5 alamethicin analog to enable EPR studies. Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate the water exposure of TOAC-alamethicin introduced into membranes of saturated or unsaturated diacyl phosphatidylcholines that were dispersed in D2O. Echo-detected EPR spectra were used to assess the degree of assembly of the peptide in the membrane, via the instantaneous diffusion from intermolecular spin-spin interactions. The profile of residue exposure to water differs between membranes of saturated and unsaturated lipids. In monounsaturated dioleoyl phosphatidylcholine, D2O-ESEEM intensities decrease from TOAC(1) to TOAC(8) and TOAC(16) but not uniformly. This is consistent with a transmembrane orientation for the protoassembled state, in which TOAC(16) is located in the bilayer leaflet opposite to that of TOAC(1) and TOAC(8). Relative to the monomer in fluid bilayers, assembled alamethicin is disposed asymmetrically about the bilayer midplane. In saturated dimyristoyl phosphatidylcholine, the D2O-ESEEM intensity is greatest for TOAC(8), indicating a more superficial location for alamethicin, which correlates with the difference in orientation between gel- and fluid-phase membranes found by conventional EPR of TOAC-alamethicin in aligned phosphatidylcholine bilayers. Increasing alamethicin/lipid ratio in saturated phosphatidylcholine shifts the profile of water exposure toward that with unsaturated lipid, consistent with proposals of a critical concentration for switching between the two different membrane-associated states.

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 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 Cu2+ or Zn2+ 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.

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. The results show that, in the absence of the alpha-helix segment, the overall protein structure is maintained, and that only the Cu site is slightly modified. In contrast, the protein stability is diminished by about 60% with respect to the wild-type azurin. Moreover, the unfolding pathway of the mutant azurin involves the presence of detectable intermediates. In comparison with previous studies concerning other small beta-sheet cupredoxins, the results as a whole support the hypothesis that the presence of the alpha-helix can switch the folding of azurin from a hierarchic to a nonhierarchic mechanism in which the highly conserved beta-sheet core provides a scaffold for cooperative folding of the wild-type protein.

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. A region with high conformational instability was found in the 'southern' end of the molecule, far away from the copper site and the binding sites for the redox partners of azurin. By excluding the 'southern' region from the subsequent analysis, correlated motions were identified in the hydrophobic patch that surrounds the protein active site. The simulation results are in excellent agreement with recent NMR data on azurin in solution [A. V Zhuravleva, D. M. Korzhnev, E. Kupce, A. S. Arseniev, M. Billeter, V. Y. Orekhov, Gated electron transfers and electron pathways in azurin: a NMR dynamic study at multiple fields and temperatures, J. Mol. Biol. 342 (2004) 1599-1611] and suggest a rationale for cooperative displacements of protein residues that are thought to be critical for the electron transfer process. A number of other structural and dynamic features of azurin are discussed in the context of the blue copper protein family and an explanation is proposed to account for the variability/conservation of some regions in the cupredoxins. (c) 2006 Elsevier B.V. All rights reserved.

Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate, $${\text{N}}_{3} \overset k \longleftrightarrow {\text{U}}_{3} \xrightarrow{k}{\text{F}} $$ (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 ≈ 85°C, i.e., 15°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 $${\text{N}}_{3} \xrightarrow{k}{\text{F}}_{3} , $$ 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.

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 D2O-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. Water penetration into the membrane is seen in the D2O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.

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.

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type I 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)approximate to 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 double left right arrow U double right arrow 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. (c) 2005 Elsevier B.V All rights reserved.

A comparative study of the thermal stability of wild type poplar plastocyanin and of a mutant form containing a disulfide bridge between residues 21 and 25 was performed using differential scanning calorimetry and optical spectroscopic techniques.For wild type plastocyanin the transition temperature, determined from the calorimetric profiles, is 62.7 degreesC at the scan rate of 60 degreesC/h, whereas for the mutant it is reduced to 58.0 degreesC. In both cases, the endothermic peak is followed by an exothermic one at higher temperatures.The unfolding process monitored by optical absorption at 596 nm also reveals a reduced thermal stability of the mutated plastocyanin compared to the wild type protein, with transition temperatures of 54.8 and 58.0 degreesC, respectively. For both proteins, the denaturation process was found to be irreversible and dependent on the scan rate preventing the thermodynamic analysis of the unfolding process.In parallel, small conformational changes between wild type and mutant plastocyanin emerge from fluorescence spectroscopy measurements. Here, a difference in the interaction of the two proteins between the microenvironment surrounding the fluorophores and the solvent was proposed.The destabilization observed in the disulfide containing mutant of plastocyanin suggests that the double mutation, Ile2lCys and Glu25Cys, introduces strain into the protein which offsets the stabilizing effect expected from the formation of a covalent crosslink. (C) 2004 Elsevier B.V. All rights reserved.

Spin-echo decays of spin-labelled phospholipids have been recorded to study the chain dynamics in the low-temperature phases of dipalmitoyl phosphatidylcholine membranes with and without 50 mol% cholesterol. The phase-memory relaxation time, T2M, depends on the position of spin-labellingin the sn-2 chain, and on the presence of cholesterol. A biphasic temperature dependence of T2M is obtained over the range 150–270 K. Echo-detected field-swept absorption EPR spectra were recorded as a function of the echo delay time, s. The echo-detected EPR lineshapes show a strongdependence on s, revealinganisotropic phase relaxation arisingfrom torsional chain motions. Cholesterol has a large effect on torsional oscillations about the chain long axis. Small-amplitude chain motions in the low-temperature phases may be important for cryopreservation of membranes. 2003 Elsevier Science (USA). All rights reserved.

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 effect of azide and thiocyanate on the structure and dynamics of wild type and disulfide bond depleted azurin and of amicyanin has been investigated by electron paramagnetic resonance (EPR) spectroscopy at low temperature. The analysis of the EPR spectra, which can be described in terms of Gaussian distributions of the components of the axial symmetric (g) over left right arrow and (A) over left right arrow tensors of the spin-Hamiltonian, has shown that the two small exogenous ligands, known as chaotropic agents, are effective in reducing the structural heterogeneity of the proteins, Such a reduction, quantified by the standard deviations sigma(gparallel to) and sigma(A\) and obtained by simulation of the experimental EPR spectra, depends on azide and thiocyanate concentration in solution. In particular, the comparison of the sigma(a\) and sigma(Aparallel to) values found for the protein samples investigated points out that the lower the protein to anion molar ratios (1:50; 1:100) are, the more marked the reduction in structural heterogeneity is. The thiocyanate effect is stronger than the azide one. Furthermore, the reduction in structural heterogeneity is more marked in the azurins than in amicyanin and the Cys3Ala/Cys26Ala azurin mutant is less flexible compared to the wild-type protein. The effect observed upon N-3(-) and SCN- addition in solution is very similar to that observed when glycerol is added to the solution, suggesting that such perturbing agents behave like cryoprotectors, affecting the protein-solvent interactions in such a way as to suppress the large amplitude motions, (C) 2002 Elsevier Science Inc. All rights reserved.

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 °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 °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.

A comparative investigation of the effects of cooling rate and solvent physicochemical properties on the structural heterogeneity of wild-type and disulfide bond depleted azurin (Cys3Ala/Cys26Ala) and of amicyanin has been performed by EPR spectroscopy and computer simulation. By describing the spectral features of the EPR spectra in terms of Gaussian distributions of the components of the $$\mathop{\bf g}\limits^{\leftrightarrow}$$ and $$\mathop{\bf A}\limits^{\leftrightarrow}$$ tensors of the spin Hamiltonian, we have shown that either the cooling rate or the solvent composition affect the structural heterogeneity of the proteins. Such a heterogeneity has been quantified by the standard deviations σg || and σA || of the parallel components of the axially symmetric tensors. In particular, both parameters become smaller after the slow cooling cycle; such a reduction is more significant when glycerol is added as co-solvent to the protein solutions. The comparison of the σg || and σA || values found, for the copper proteins investigated, highlights that the reduction is more marked in the azurins compared to amicyanin and that the Cys3Ala/Cys26Ala azurin mutant has a structural heterogeneity lower than that shown by the wild-type protein. The remarkable similarity of the copper coordination sphere of the proteins suggests a more rigid structure of the azurin protein matrix in the absence of the disulfide bridge compared to wild-type azurin and of amicyanin with respect to both forms of azurin. The former result establishes an important role for the -SS- bond in modulating the flexibility of wild-type azurin.

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 (La Rosa et al. 1995. 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 protein's 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 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 Delta Cp, were used to calculate the thermodynamic stability, Delta G, 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.

The self-assembly of proteins and polypeptides in amyloid fibrillar aggregates is rapidly emerging as a promising route towards the fabrication of nano-objects with controlled morphologies and properties. Transition metal ions are known to play an important but elusive role in the amyloid fibrillation associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We have considered the effect of copper ions Cu2+ on the nanoscale morphology and fibrillation kinetics of beta-lactoglobulin (beta LG), a model protein not related to amyloid diseases, which denatures and self-assembles in nanofibrils upon heating at low pH and ionic strength. We found that an increasing level of Cu2+ decreases the enthalpy of denaturation and significantly increases the rate of fibril nucleation, also producing a small increase of the fibril elongation rate. Cu2+ acts as a catalytic agent during protein denaturation and fibrillation, without binding to beta LG before or after heating, and produces only minor changes in fibril morphology. Beside possible implications for amyloid pathologies in vivo, our results suggest that transition metal ions can be used to control the self-assembly of protein-based nano-objects in vitro.

The structural organization and overall stability of biological systems is largely determined by the interaction existing between their components and between these components and the aqueous environment, i.e., the solvent. As concerns the latter interaction, there is convincing evidence that the behaviour of membrane components and proteins changes by changing the physico-chemical properties (pH, ionic strength, polarity) of the solvent. In fact, it has been shown by x-ray diffraction and calorimetric investigations that the basic structural arrangement of biological membrane, i.e., the lipid bilayer, in presence of various compounds such as glycerol [1–4], small surface active molecules [5], alcohols [3, 6–9] and ions [10] above a threshold concentration, results completely interdigitated in the gel phase of saturated, symmetrical chain phosphatidylcholines. In this phase, the opposing lipid monolayers interpenetrate each other so that the terminal methyl groups of the lipid chains are located near the interfacial region on the opposite side of the lipid lamellae [for a review see ref. 11].

Transitions between conformational substates of membrane proteins can be driven by torsional librations in the protein that may be coupled to librational fluctuations of the lipid chains. Here, librational motion of spin-labeled lipid chains in membranous Na,K-ATPase is investigated by spin-echo electron paramagnetic resonance. Lipids at the protein interface are targeted by using negatively charged spin-labeled fatty acids that display selectivity of interaction with the Na,K-ATPase. Echo-detected electron paramagnetic resonance spectra from native membranes are corrected for the contribution from the bilayer regions of the membrane by using spectra from dispersions of the extracted membrane lipids. Lipid librations at the protein interface have a flat profile with chain position, whereas librational fluctuations of the bilayer lipids increase pronouncedly from C-9 onward, then flatten off toward the terminal methyl end of the chains. This difference is accounted for by increased torsional amplitude at the chain ends in bilayers, while the amplitude remains restricted throughout the chain at the protein interface with a limited lengthening in correlation time. The temperature dependence of chain librations at the protein interface strongly resembles that of the spin-labeled protein side chains, suggesting solvent-mediated transitions in the protein are driven by fluctuations in the lipid environment.

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. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2τ. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.

The dynamics of spin-labeled lipid chains in the low-temperature phases of dipalmitoyl phosphatidylcholine (DPPC) membranes, with and without equimolar cholesterol, have been investigated by pulsed electron paramagnetic resonance (EPR) spectroscopy. Echo-detected spectra from the two-pulse, primary spin-echo (pulse sequence: π/2-τ-π-τ-echo) are used to detect rapid angular motions, on the time scale of the phase memory time (T 2 M ) that is in the nanosecond regime. Echo-detected spectra from the three-pulse, stimulated spin-echo (pulse sequence: π/2-τ-π/2-T-π/2-τ-echo) are used to detect slow angular motions, on the time scale of the spin-lattice relaxation time (T 1 ) that is in the microsecond regime. Spectra recorded at very low temperature (77 K) are used to correct the two-pulse echo spectra for instantaneous diffusion that arises from dipolar spin-spin interactions between different spin labels. Echo-detected inversion recovery spectra are used to correct the three-pulse echo spectra for intrinsic spin-lattice relaxation and large-scale spectral diffusion induced by nitrogen nuclear spin flips. The dependence of the echo-detected spectral line shapes on the two time delays, r and T, can be simulated adequately by using a simple two-state model to represent the small-amplitude librational motions in the low-temperature membrane phases. The fast librational motion has isotropic character, no singly defined direction of the librational axis, and is segmental in nature, depending on chain position and also on the presence of cholesterol. The slow librational motion is of a more global, cooperative nature, being independent of chain position and cholesterol content.

The thermal denaturation of azurin from Pseudomonas aeruginosa was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR), and optical density (OD) experiments, with the aim of determining its thermodynamic stability and the thermally induced conformational changes of its active site. DSC experiments have shown an irreversible and complex unfolding path. In order to characterize the kinetically controlled step, DSC measurements were carried out at different scan rates. An extrapolation of the experimental heat capacity data to infinite scan rate allowed all the kinetic and thermodynamic parameters related to the process to be obtained. All these parameters extracted from the calorimetric data were verified by means of a curve-fitting program using an equation containing all information necessary to fully describe the unfolding process in details. Thermal denaturation, followed up to 82 degrees C by ESR and OD measurements, allowed us to study the structural variations of the copper environment at different temperatures. The Delta H-U thermodynamic, together with the value of Delta C-p calculated according to an approach taking into account the common features of protein unfolding and dissolution of hydrophobic compounds, was used to evaluate the thermodynamic stability (Delta G) for the reversible process over the entire temperature range of denaturation. The high value of the maximum stability thus calculated was explained by the stabilizing effect of copper.

The association of water (D2O) with phospholipid membranes was studied by using pulsed-electron spin resonance techniques. We measured the deuterium electron spin echo modulation of spin-labeled phospholipids by D2O in membranes of dipalmitoyl phosphatidylcholine with and without 50 mol% of cholesterol. The Fourier transform of the relaxation-corrected two-pulse echo decay curve reveals peaks, at one and two times the deuterium NMR frequency, that arise from the dipolar hyperfine interaction of the deuterium nucleus with the unpaired electron spin of the nitroxide-labeled lipid. For phosphatidylcholine spin-labeled at different positions down the sn-2 chain, the amplitude of the deuterium signal decreases toward the center of the membrane, and is reduced to zero from the C-12atom position onward. At chain positions C-5 and C-7 closer to the phospholipid headgroups, the amplitude of the deuterium signal is greater in the presence of cholesterol than in its absence. These results are in good agreement with more indirect measurements of the transmembrane polarity profile that are based on the 14N-hyperfine splittings in the conventional continuous-wave electron spin resonance spectrum.