Conformational heterogeneity and spin-labeled -SH groups: pulsed EPR of Na,K-ATPase.

Membranous Na,K-ATPase from shark salt gland and from pig kidney was spin-labeled on class I -SH groups in the presence of glycerol, or on class II -SH groups in the absence of glycerol. The class I-labeled preparations retain full enzymatic activity, whereas the class II-labeled preparations are at least partially inactivated. This provides an excellent testbed on which to demonstrate how advanced electron paramagnetic resonance (EPR) can provide novel information on specific residues in unique environments in a complex, membrane-bound transport system. The polarity of the environment, and the librational dynamics and conformational exchange, of the spin-labeled groups were studied with pulsed EPR by using electron spin echo envelope modulation (ESEEM) spectroscopy and spin-echo detected (ED) EPR spectroscopy, respectively. 2H-ESEEM spectra of membranes dispersed in D2O reveal that class I groups of the shark enzyme are more exposed to water than are those of the pig enzyme or class II groups of either species, consistent with the more superficial membrane location in the former case. Spin-echo decay curves indicate conformational heterogeneity at low temperatures (<150 K), but a more homogeneous conformational state at higher temperatures that is characterized by a single phase-memory T2M relaxation time. Conventional EPR lineshapes also demonstrate conformational microheterogeneity at low temperatures: the inhomogeneously broadened lines narrow progressively with increasing temperature reaching an almost pure Lorentzian line shape at temperatures of ca. 220 K and above. The inhomogeneous broadening at low temperature is well described by a Gaussian distribution of Lorentzian lines. ED spectra as a function of echo-delay time demonstrate the onset of rapid librational motions of appreciable amplitude, and slower conformational exchange, at temperatures above 220 K. These motions could drive transitions between the different conformational substates, which are frozen in at lower temperatures but contribute to the pathways between the principal enzymatic intermediates at higher temperatures.

Intramembrane water associated with TOAC spin-labeled alamethicin: electron spin-echo envelope modulation by D2O.

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.

Thermally induced denaturation and aggregation of BLG-A: effect of the Cu(2+) and Zn (2+) metal ions.

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 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.

Bipolar tetraether lipids: chain flexibility and membrane polarity gradients from spin-label electron spin resonance.

Membranes of thermophilic Archaea are composed of unique tetraether lipids in which C40, saturated, methyl-branched biphytanyl chains are linked at both ends to polar groups. In this paper, membranes composed of bipolar lipids P2 extracted from the acidothermophile archaeon Sulfolobus solfataricus are studied. The biophysical basis for the membrane formation and thermal stability is investigated by using electron spin resonance (ESR) of spin-labeled lipids. Spectral anisotropy and isotropic hyperfine couplings are used to determine the chain flexibility and polarity gradients, respectively. For comparison, similar measurements have been carried out on aqueous dispersions of diacyl reference lipid dipalmitoyl phosphatidylcholine and also of diphytanoyl phosphatidylcholine, which has methyl-branched chains. At a given temperature, the bolaform lipid chains are more ordered and less flexible than in normal bilayer membranes. Only at elevated temperatures (80 degrees C) does the flexibility of the chain environment in tetraether lipid assemblies approach that of fluid bilayer membranes. The height of the hydrophobic barrier formed by a monolayer of archaebacterial lipids is similar to that in conventional fluid bilayer membranes, and the permeability barrier width is comparable to that formed by a bilayer of C16 lipid chains. At a mole ratio of 1:2, the tetraether P2 lipids mix well with dipalmitoyl phosphatidylcholine lipids and stabilize conventional bilayer membranes. The biological as well as the biotechnological relevance of the results is discussed.

The effect of copper/zinc replacement on the folding free energy of wild type and Cys3Ala/Cys26Ala azurin.

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.

Effects of chaotropic anions on the distribution of conformational substates of amicyanin, wild type and Cys3Ala/Cys26Ala azurin mutant.

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 and <--> A 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(g axially) and sigma(A axially) and obtained by simulation of the experimental EPR spectra, depends on azide and thiocyanate concentration in solution. In particular, the comparison of the sigma(g axially) and sigma(A axially) 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.

Evidence of reduced flexibility in disulfide bridge-depleted azurin: a molecular dynamics simulation study.

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.

Lipid chain length effect on the phase behaviour of PCs/PEG:2000-PEs mixtures. A spin label electron spin resonance and spectrophotometric study.

Spin-label electron spin resonance (ESR) spectroscopy and spectrophotometry at fixed wavelength are used to study fully hydrated aqueous dispersions of phosphatidylcholines (PCs) with poly(ethylene glycol:2000)-phosphatidylethanolamines (PEG:2000-PEs). PEG:2000-PE is a micelle-forming polymer-lipid that is extensively used for increasing the lifetime of PC liposomes in the blood circulation through a steric stabilisation effect. The PC lipids and the PEG:2000-PE polymer-lipids have the same acyl chain length of either dimiristoyl (DM) or distearoyl (DS) chains. DMPC/PEG:2000-DMPE and DSPC/PEG:2000-DSPE mixtures were investigated over the entire range of relative compositions (0-100 mol%). In both dispersions, the low-temperature conventional spin label ESR spectra and the temperature dependence of the absorbance at 400 nm give an indication of the conversion from lamellae to micelles with increasing PEG:2000-PEs content. The physical state of the lipid assemblies, lamellar or micellar, is dependent not only on PEG:2000-PEs content, but also on the length of hydrocarbon chain of the lipid matrix. Micellisation is attained more readily in dispersions with longer hydrocarbon chains (i.e. in DSPC/PEG:2000-DSPE mixtures) than in those with shorter acyl chains (i.e. in DMPC/PEG:2000-DMPE mixtures). Saturation transfer ESR (ST-ESR) and absorbance measurements reflect the disaggregation of the bilayers and a reduction in the size of the lipid aggregates by PEG:2000-PEs at low content.

Structural heterogeneity of blue copper proteins: an EPR study of amicyanin and of wild-type and Cys3Ala/Cys26Ala mutant azurin.

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 g and A 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 sigmag and sigmaA 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 cosolvent to the protein solutions. The comparison of the deltag and sigmaA 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.

Lipid membrane expansion and micelle formation by polymer-grafted lipids: scaling with polymer length studied by spin-label electron spin resonance.

Spin-label electron spin resonance (ESR) spectroscopy and auxiliary optical density measurements are used to study lipid dispersions of N-poly(ethylene glycol)-dipalmitoyl phosphatidylethanolamine (PEG:5000-DPPE) mixed with dipalmitoyl phosphatidylcholine (DPPC). PEG:5000-DPPE bears a large hydrophilic polymer headgroup (with approximately 114 oxyethylene monomers) and is commonly used for steric stabilization of liposomes used in drug delivery. Comparison is made with results from mixtures of DPPC with polymer lipids bearing shorter headgroups (approximately 45 and 8 oxyethylene monomers). ESR spectra of phosphatidylcholine spin-labeled on the 5-C atom position of the sn-2 chain are shown to reflect the area expansion of the lipid membranes by the lateral pressure exerted in the polymer brush, in a way that is consistent with theory. The lipid chain packing density at the onset of micelle formation is the same for all three PEG-lipids, although the mole fraction at which this occurs differs greatly. The mole fraction at onset scales inversely with the size of the polymer headgroup, where the experimental exponent of 0.7 is close to theoretical predictions (viz. 0.55-0.6). The mole fraction of PEG-lipid at completion of micelle formation is more weakly dependent on polymer size, which conforms with theoretical predictions. At high mole fractions of PEG:5000-DPPE the dependence of lipid packing density on mole fraction is multiphasic, which differs qualitatively from the monotonic decrease in packing density found with the shorter polymer lipids. Lipid spin-label ESR is an experimental tool that complements theoretical analysis using polymer models combined with the lipid equation of state.

Molecular and mesoscopic properties of hydrophilic polymer-grafted phospholipids mixed with phosphatidylcholine in aqueous dispersion: interaction of dipalmitoyl N-poly(ethylene glycol)phosphatidylethanolamine with dipalmitoylphosphatidylcholine studied by spectrophotometry and spin-label electron spin resonance.

Spin-label electron spin resonance (ESR) spectroscopy, together with optical density measurements, has been used to investigate, at both the molecular and supramolecular levels, the interactions of N-poly(ethylene glycol)-phosphatidylethanolamines (PEG-PE) with phosphatidylcholine (PC) in aqueous dispersions. PEG-PEs are micelle-forming hydrophilic polymer-grafted lipids that are used extensively for steric stabilization of PC liposomes to increase their lifetimes in the blood circulation. All lipids had dipalmitoyl (C16:0) chains, and the polymer polar group of the PEG-PE lipids had a mean molecular mass of either 350 or 2000 Da. PC/PEG-PE mixtures were investigated over the entire range of relative compositions. Spin-label ESR was used quantitatively to investigate bilayer-micelle conversion with increasing PEG-PE content by measurements at temperatures for which the bilayer membrane component of the mixture was in the gel phase. Both saturation transfer ESR and optical density measurements were used to obtain information on the dependence of lipid aggregate size on PEG-PE content. It is found that the stable state of lipid aggregation is strongly dependent not only on PEG-PE content but also on the size of the hydrophilic polar group. These biophysical properties may be used for optimized design of sterically stabilized liposomes.

A spectroscopic and calorimetric investigation on the thermal stability of the Cys3Ala/Cys26Ala azurin mutant.

The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the 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.

Sterically stabilized liposomes of DPPC/DPPE-PEG:2000. A spin label ESR and spectrophotometric study.

The chain dynamics and the thermotropic phase behavior of sterically stabilized liposomes obtained introducing in the host bilayer matrix of DPPC up to 7 mol% of the polymer-lipid DPPE-PEG:2000 were investigated by spin label electron spin resonance spectroscopy and spectrophotometry. The experimental data indicate that the dispersions have the dynamic and thermotropic characteristics of normal lamellar phase. Moreover, using spin labels that locate both in the interfacial and in the hydrocarbon regions, namely TEMPO-stearate, 5- and 16-PCSL, we find that relative to the unmodified DPPC bilayers, the polymer-grafted bilayers are loosely packed in the interfacial region and have reduced chain mobility in the gel phase. From the temperature dependence of the partition coefficient (P(c)), of the spin probe DTBN between the aqueous and the fluid hydrophobic regions of the bilayers and from the melting curves of the absorbance at 400 nm, we observe a slight influence on the endothermic phase transitions when increasing the concentration of the polymer-lipid in the DPPC bilayers, the influence being more evident in the pre-transition.

An EPR investigation on the structural heterogeneity in copper azurin and plastocyanin.

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.

Spin label EPR study of the effects of monovalent cations, anions, and chaotropics on DPPC multilayers.

The electron paramagnetic resonance (EPR) spectroscopy with the spin-labeling technique is used to investigate the effects of monovalent ions on multibilayer dispersions of dipalmitoylphosphatidylcholine (DPPC). Cations of chloride salt (Li+, Na+, K+ and Cs+) and anions of potassium salt (Br-, Cl- and NO3-) at the concentration of 1 M do not affect both the molecular order and the packing of the phospholipid acyl chains in the different phases compared to DPPC dispersions in buffer. Moreover, they leave unaffected the characteristics of the main transition, whereas the pre-transition temperature increases of about 2 degrees C in the presence of cations and changes in the order NO3- < Br- < buffer < Cl- in the presence of anions. The anions that exhibit pronounced chaotropic properties (I-, SCN-) result the most effective in perturbing the bilayer. In fact, DPPC dispersions in 1 M of these salt solutions do not show the pre-transition and have the main one shifted to lower temperature in the order: SCN- < 1- < buffer. Furthermore, the spin-label EPR results on the lipid chain dynamics indicate the presence of a flexibility gradient both in DPPC/buffer and in DPPC/chaotropic systems. However, the chaotropic anions influence the DPPC hydrocarbon chains in the gel phase in a manner such that interpenetration or interdigitation of the terminal methyl groups from opposing monolayers is likely to occur.

Chlorofluorocarbon interaction with DPPC vesicles: an ESR investigation.

The effect of the chlorofluorocarbon halotane on DPPC unilamellar vesicles was investigated by Electron Spin Resonance (ESR) spectroscopy. The X-band ESR spectra showed prominent thermotropic behaviour of the interfacial region of vesicles at different halotane concentrations. A model of molecular interaction between phospholipids and chlorofluorocarbon is also proposed.

Thermal behaviour of lymphocyte membrane: ESR investigation.

The order parameter, S, of the plasma membrane of in toto human peripheral blood lymphocytes was obtained by electron spin resonance spectroscopy in the temperature range 25-41 degrees C. This membrane is completely in the liquid crystalline state above 31 degrees C. In presence of the antigen ETB from Staphylococcus aureus at the concentration of 4 micrograms/3 x 10(7) cells an overall decrease of the order parameter for this membrane is observed. The decrease of S is followed by an upwards shift at about 35 degrees C of the temperature of the liquid crystalline state.