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.

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.

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.

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.

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.

Lipid chain motion in an interdigitated gel phase: conventional and saturation transfer ESR of spin-labeled lipids in dipalmitoylphosphatidylcholine-glycerol dispersions.

The lipid chain dynamics in the interdigitated gel phase of dipalmitoylphosphatidylcholine (DPPC) dispersed in glycerol and in the fully hydrated noninterdigitated gel phase in aqueous buffer were compared by using conventional and saturation transfer electron spin resonance (ESR) spectroscopy. Twelve different positional isomers of phosphatidylcholine spin-labeled in the sn-2 chain were used to characterize the chain motion. The outer hyperfine splittings of the conventional ESR spectra and the line height ratios at the diagnostic spectral positions in the saturation transfer ESR spectra were taken as indices of the rotational mobility of the labeled chain segments in the gel phase (0-40 degrees C). The conventional spin label ESR spectra revealed a gradient of increasing mobility on proceeding down the chain toward the terminal methyl end in the fully hydrated DPPC gel phase bilayer structure. This gradient was absent in the interdigitated gel phase, i.e., the rotational mobility throughout the length of the lipid chain was comparable to that near the polar interface, on the conventional ESR time scale. Values of the outer hyperfine splitting for spin labels at the 5- and 14-C atom positions in the chain were 65.5 and 61.0 G in buffer, respectively, and 67.0 G for both positions in glycerol, at 0 degrees C. At 35 degrees C, still in the gel phase, these differences between the two systems were much greater. Saturation transfer ESR measurements revealed that the motion throughout the chain was restricted on the microsecond time scale in the interdigitated phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and dynamics of annealing during sub-gel phase formation in phospholipid bilayers: A saturation transfer electron spin resonance study.

The saturation transfer electron spin resonance (STESR) spectra of spin-labeled phosphatidylcholine have been used to follow the kinetics of conversion from the gel phase to the sub-gel phase in aqueous bilayers of dipalmitoyl phosphatidylcholine. This is a simple, well-defined model system for lipid domain formation in membranes. The integrated intensity of the STESR spectrum from the chain-labeled lipid first increases and then decreases with time of incubation in the gel phase at 0 degrees C. The first, more rapid phase of the kinetics is attributed to the conversion of germ nuclei to growth nuclei of the sub-gel phase. The increase in STESR intensity corresponds to the reduction in chain mobility of spin labels located in the gel phase at the boundaries of the growth nuclei and correlates with the increase in the diagnostic STESR line height ratios over this time range. The second, slower phase of the kinetics is attributed to growth of the domains of the sub-gel phase. The decrease in STESR intensity over this time regime corresponds to exclusion of the spin-labeled lipids from the tightly packed sub-gel phase and correlates quantitatively with calibrations of the spin label concentration dependence of the STESR intensity in the gel phase. The kinetics of formation of the sub-gel phase are consistent with the classical model for domain formation and growth. At 0 degrees C, the half-time for conversion of germ nuclei to growth nuclei is approximately 7.7 h and domain growth of the sub-gel phase is characterized by a rate constant of 0.025 h(-1). The temperature dependence of the STESR spectra from samples annealed at 0 degrees C suggests that the subtransition takes place via dissolution of sub-gel phase domains, possibly accompanied by domain fission.

Distance measurements using paramagnetic ion-induced relaxation in the saturation transfer electron spin resonance of spin-labeled biomolecules: Application to phospholipid bilayers and interdigitated gel phases.

The saturation transfer electron spin resonance (STESR) spectra of spin-labeled phosphatidylcholines in gel phase lipid bilayers are shown to be sensitive to dipolar spin-spin interactions with paramagnetic ions in the aqueous phase. The reciprocal integrated intensity of the STESR spectrum is linearly dependent on aqueous Ni(2+) ion concentration, hence, confirming the expectation that the STESR intensity is directly proportional to the spin-lattice relaxation time of the spin label. The gradient of the relaxation rate with respect to Ni(2+) ion concentration decreases strongly with the position of the nitroxide group down the sn-2 chain of the spin-labeled lipid and is consistent with a 1/R(3) dependence on the distance, R, from the bilayer surface. The values derived for the dimensions of the bilayer and lipid molecules in the case of dipalmitoyl phosphatidylcholine (DPPC) are in good agreement with those available from x-ray diffraction studies. Allowance for the multibilayer nature of the DPPC dispersions gives an estimate of the water layer thickness that is also consistent with results from x-ray diffraction. The profile of the paramagnetic ion-induced relaxation is drastically changed with DPPC dispersions in glycerol for which the lipid chains are known to be interdigitated in the gel phase. The terminal methyl groups of the lipid chains are located approximately in register with the C-3 atoms of the sn-2 chain of the oppositely oriented lipid molecules in the interdigitated phase. The thickness of the lipid layer and the effective thickness of the lipid polar group are reduced by approximately 40% in the interdigitated phase as compared with the bilayer phase. The calibrations of the distance dependence established by use of spin labels at defined chain positions should be applicable to STESR measurements on other biological systems.

Effect of high electrolyte concentration on the phase transition behaviour of DPPC vesicles: a spin label study.

We have investigated by Electron Spin Resonance spectroscopy, the effects of high electrolyte concentration on the phase transitions of unilamellar vesicles of dipalmitoylphosphatidylcholine at the pH values of 5.0 and 9.0. Using the 5-nitroxide stearic acid as spin probe we have found that, at both pH values, the lipid main phase transition is not quite affected by variations of the electrolyte concentration up to the value of 3 M. Instead, the pretransition at pH 5.0 disappears in the presence of 1 M electrolyte, and at pH 9.0, the pretransition temperature shifts upward from 25.5 to 31.0 degrees C when the electrolyte concentration reaches the value of 3 M. The observed results on the pre- and main phase transition widths, transition temperatures and their cooperativity indicate that the presence of salt in the bulk solution leads to structural changes of the lipid bilayer which essentially concern either the polar zone or the hydrogen belt region of the DPPC vesicles. The extent of observed perturbation depends on salt concentration.

Effect of inhalation anesthetics on spin-labeled cholesterol containing DPPC vesicles.

We have investigated by means of electron spin resonance (ESR) spectroscopy the influence of three inhalation anesthetics, i.e. halothane, chloroform and diethyl ether, on the interfacial and hydrophobic region as well of 38 mol% cholesterol containing DPPC unilamellar vesicles. The study has been carried out in the temperature range 25-45 degrees C. The variation of the order parameter, S, vs temperature of the lipid phase indicates that with this content of cholesterol the characteristic gel----liquid crystalline main phase transition of DPPC, normally occurring at Tt approximately 41 degrees C, disappears. When halothane and chloroform are added to the vesicles suspension up to [DPPC]/[anesthetic] molar ratio of 1:1 the main phase transition, as detected with the stearic acid spin label I (12,3), reappears again and it results down shifted at Tt approximately 35 and 39 degrees C, respectively. In presence of diethyl ether, instead, the main phase transition is not observable also at the highest concentration of anesthetic used. Moreover, halothane and chloroform affect similarly the hydrophobic core of cholesterol + DPPC vesicles which, in turn, results to be different from the action exerted by diethyl ether in the same region. The ESR findings are discussed in terms of competitive effects shown by cholesterol and inhalation anesthetics. Moreover, the interfacial region of CHOL + DPPC vesicles results to be the target of anesthetics.