Spin-label electron spin resonance studies on the interactions of lysine peptides with phospholipid membranes.

The interactions of lysine oligopeptides with dimyristoyl phosphatidylglycerol (DMPG) bilayer membranes were studied using spin-labeled lipids and electron spin resonance spectroscopy. Tetralysine and pentalysine were chosen as models for the basic amino acid clusters found in a variety of cytoplasmic membrane-associating proteins, and polylysine was chosen as representative of highly basic peripherally bound proteins. A greater motional restriction of the lipid chains was found with increasing length of the peptide, while the saturation ratio of lipids per peptide was lower for the shorter peptides. In DMPG and dimyristoylphosphatidylserine host membranes, the perturbation of the lipid chain mobility by polylysine was greater for negatively charged spin-labeled lipids than for zwitterionic lipids, but for the shorter lysine peptides these differences were smaller. In mixed bilayers composed of DMPG and dimyristoylphosphatidylcholine, little difference was found in selectivity between spin-labeled phospholipid species on binding pentalysine. Surface binding of the basic lysine peptides strongly reduced the interfacial pK of spin-labeled fatty acid incorporated into the DMPG bilayers, to a greater extent for polylysine than for tetralysine or pentalysine at saturation. The results are consistent with a predominantly electrostatic interaction with the shorter lysine peptides, but with a closer surface association with the longer polylysine peptide.     

Spin-label studies of tropomyosin.

Studies are reported on nitroxide spin-labeled tropomyosin. The labels attach to sulfhydryl groups and to amino groups. The amino spins are highly mobile, the sulfhydryl much less so. Spin count studies show an average of approximately 0.5 labeled sulfhydryl/tropomyosin molecule and only approximately 0.15 labeled amino group/molecule. The spectra are used tostudy the denaturation of tropomyosin by guanidine hydrochloride. The information obtained reveals the course of denaturation at sites near the sulfhydryl group. It is found that these sites are more susceptible to guanidine than the bulk of the molecule; denaturation at the sulfhydryl sites is complete by 1.5 M guanidine, whereas optical studies indicate the molecule as a whole is not completely denatured until the concentration reaches 3.5 M. Spectra are also shown of tropomyosin fibers oriented variously with respect to the applied magnetic field. Strong orientation effects are seen and these indicate that the sulfhydryl-attached spins (but not the amino-attached spins) have a definite orientation in the fiber. Interpretation of the spectra reveals that the normal to the nitroxide plane is inclined to the fiber axis at an angle of 50 degrees. Circular dichroism studies in the tyrosine region also reveal drastic changes with guanidine denaturation, confirming the idea that denaturation produces pronounced increase in mobility at the beta carbon (as in the sulfhydryl casey). A strong negative band existing only in helical tropomyosin at pH's where the tyrosines are uncharged appears to be due to interaction of tyrosines with the helical backbone, whereas the appearance of a strong positive CD band at 250 nm at high pH (approximately11) seems to be ascribable to interaction between the charged phenolic groups and the dissymmetric backbone alpha-carbon atom.     

Spin-label studies of the lipid regions of spinach thylakoids and a detergent-derived oxygen-evolving Photosystem II preparation

We have compared the fluidity of thylakoid membranes with the membrane present in a Triton X-100-derived, oxygen-evolving Photosystem II (PS II) preparation using two different spin labels. Data obtained with 2,2,6,6-tetramethylpipiridine-N-oxyl (TEMPO) shows that the PS II preparation contains less fluid membrane than the thylakoid. The TEMPO partition parameter (f) is about 2.5-times greater for the thylakoids at 6 mg chlorophyll/ml than for the PS II preparation at the same chlorophyll concentration. Similarly, the rotational correlation time, τ, of TEMPO residing in the membrane of the PS II preparation is about 2-times longer than the τ for TEMPO in the thylakoid membrane. A spin label which partitions more completely into the bilayer, 2-heptyl-2-hexyl-5,5-dimethyloxazolidine-N-oxyl (7N14), indicates a much greater fluidity in the thylakoid membrane than the membrane of the PS II preparation. The PS II preparation appears to have a hydrocarbon phase which approaches the rigid limit of EPR detectable motion. These results are discussed in terms of possible lipid depletion in the PS II preparation and in terms of lateral heterogeneity of hydrocarbon fluidity in the thylakoid membrane caused by the lateral heterogeneity in protein components.     

Pretranstional phenomena in phospholipid/water multilayers.

We have measured the water order in monodomain phospholipid samples using 2H nuclear magnetic resonance (NMR) and analyzed the splittings in terms of critical exponents. Our data and the model developed to interpret them in terms of fluctuations provide an explanation of the puzzling sharp reduction of water order near the chain-ordering phase transition. The temperature range of the fluctuations is approximately the same as that observed for increased 22Na+ efflux from phospholipid vesicles.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells.

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 +/- 1.14 . 10(5) and (2.2 +/- 1.2) . 10(5) spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethylmaleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

ESR studies on the orientation of cholesteryl ester in phosphatidylcholine multilayers.

The alignment of cholesteryl esters in multilayer phosphatidylcholine membranes was investigated using two spin-labelled cholesteryl esters: 10 : 3 ester (I) and 1 : 14 ester (II). The nitroxide label of I is aligned in the membrane with a very large angle of tilt (47 degrees +/- 1.5 degrees) with respect to the normal to the membrane surface; II does not show such a tilt. I gives spectra corresponding to immobilized label while II gives nearly isotropic spectra. Ascorbate treatment of the multilayers shows that the labels in I and II are not present at the phosphatidylcholine-water interphase. The data supports a 'horseshoe' configuration for the cholesteryl ester in the bilayer, with both the fatty acid chain and the cholesteryl moiety extending deep into the hydrophobic region of the membrane and with the ester linkage near the surface.     

Spin-label electron paramagnetic resonance studies on the interaction of avidin with dimyristoyl-phosphatidylglycerol membranes

The interaction of avidin--a basic protein from hen egg-white--with dimyristoyl-phosphatidylglycerol membranes was investigated by spin-label electron paramagnetic resonance spectroscopy. Phosphatidylcholines, bearing the nitroxide spin label at different positions along the sn-2 acyl chain of the lipid were used to investigate the effect of protein binding on the lipid chain-melting phase transition and acyl chain dynamics. Binding of the protein at saturating levels results in abolition of the chain-melting phase transition of the lipid and accompanying perturbation of the lipid acyl chain mobility. In the fluid phase region, the outer hyperfine splitting increases for all phosphatidylcholine spin-label positional isomers, indicating that the chain mobility is decreased by binding avidin. However, there was no evidence for direct interaction of the protein with the lipid acyl chains, clearly indicating that the protein does not penetrate the hydrophobic interior of the membrane. Selectivity experiments with different spin-labelled lipid probes indicate that avidin exhibits a preference for negatively charged lipid species, although all spin-labelled lipid species indirectly sense the protein binding. The interaction with negatively charged lipids is relevant to the use of avidin in applications such as the ultrastructural localization of biotinylated lipids in histochemical studies.     

Spin-label studies of glycogen phosphorylase hosted in microemulsion droplets

ESR spectra of Phosphorylase b labelled with two different paramagnetic probes were recorded in microemulsions of various compositions, formulated with AOT and isooctane. Measurements of the hyperfine coupling constant aN and of the degree of anisotropy H1/H0, showed that: i) the polarity of the microenvironment of both spin-labels bound to the enzyme, expressed in terms of aN, is increased with increasing the hydration ratio of the reversed micelles, R; ii) a transition of the polarity between bound and free water is observed at R = 10; iii) the mobilities of the spin-labelled moieties of the enzyme are different, depending on the hydrophobicity of the site where the probe is bound and on the size of the microdroplets.     

Spin-label ESR studies of lipid-protein interactions in thylakoid membranes

Lipid-protein interactions in thylakoid membranes, and in the subthylakoid membrane fractions containing either photosystem 1 or photosystem 2, have been studied by using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted membrane lipids interacting directly with the integral membrane proteins. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with the membrane proteins and to determine the selectivity between the different lipid classes for the lipid-protein interaction. The fractions of motionally restricted lipid in the thylakoid membrane are 0.36, 0.39, and 0.53, for the spin-labeled monogalactosyldiacylglycerol, phosphatidylcholine, the phosphatidylglycerol, respectively. Spin-labeled monogalactosyldiacylglycerol exhibits very little preferential interaction over phosphatidylchline, which suggests that part of the role of monogalactosyldiacylglycerol in thylakoid membranes is structural, as is the case for phosphatidylcholine in mammalian membranes. Spin-labeled phosphatidylglycerol shows a preferential interaction over the corresponding monogalactosyldiacylglycerol and phosphatidylcholine analogues, in contrast to the common behavior of this lipid in mammalian systems. This pattern of lipid selectivity is preserved in both the photosystem 1 and photosystem 2 enriched subthylakoid membrane fractions.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Spin-label studies on the specificity of interaction of cardiolipin with beef heart cytochrome oxidase

The selectivity of interaction of various cardiolipin analogues with beef heart cytochrome oxidase in reconstituted complexes with dimyristoylphosphatidylcholine has been studied by electron spin resonance spectroscopy, using lipids spin-labeled in the acyl chains. No difference in selectivity is observed between cardiolipin and its monolyso derivative, and similarly no selectivity is observed between phosphatidylcholine and lysophosphatidylcholine. Removal of the cardiolipin charge by methylation of the phosphate groups reduces but does not eliminate selectivity relative to phosphatidylcholine. The dependence of the lipid selectivity on head group and chain composition is in the order cardiolipin approximately equal to monolysocardiolipin greater than acylcardiolipin greater than dimethylcardiolipin greater than phosphatidylcholine approximately equal to lysophosphatidylcholine, where acylcardiolipin has the spin-label chain attached at the center -OH of the head group. The degree of association of the negatively charged cardiolipin derivatives with cytochrome oxidase decreases with increasing salt concentration, to a level comparable to that for dimethylcardiolipin. At high ionic strength there is still a marked selectivity relative to phosphatidylcholine. Li+ ions are more effective in screening the interaction than are Na+ ions, and divalent ions are more effective than monovalent ions. The selectivity for cardiolipin is only slightly reduced on titrating the protein to high pH. Alkylation of the protein with N-ethylmaleimide has little effect on the titration behavior. Covalent modification of the protein by reaction with citraconic anhydride decreases the selectivity of interaction with cardiolipin. It is concluded that cardiolipin possesses an additional specificity of interaction with cytochrome oxidase other than that of purely electrostatic origin.     

Spin-label studies on the sulfhydryl environment in bovine plasma albumin. 2. The neutral transition and the A isomer.

Since we were able to demonstrate that the sulfhydryl group is located in the crevice which opens during the N--F transition (Cornell, C. N., & Kaplan, L. J. (1978) Biochemistry 17 (preceding paper in this issue), the investigation was extended by characterizing the environment during the N--B transition and in the A isomer. The results indicate that the N--B and N--F transitions are very similar in that the sulfhydryl group moves from a restricted to unhindered environment during both. The use of molecular dipstick technique further demonstrated the similarity between the F and A forms. However, since A is a covalently stabilized form of albumin after a pH dependent transition, it retains its properties during subsequent pH changes rather than reverting to the N form. We were thus able to titrate spin-labeled A through the pH range of the acidic transitions without detecting the N--F transition. Isoelectric focusing analysis of A generated during alkaline aging and purified by SP-Sephadex chromatography indicates that it is a mixture of a small number of albumin forms rather than the large number of components once thought to be formed during aging.     

Spin-label studies of lipid-protein interactions with reconstituted band 3, the human erythrocyte chloride-bicarbonate exchanger.

Lipid-protein interactions in reconstituted band 3 preparations were investigated by using spin-labeled lipids in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Purified erythrocyte band 3 was reconstituted into egg phosphatidylcholine liposomes at high protein density with preservation predominantly of the dimeric state. Lipid-protein associations were revealed by the presence of a component in the EPR spectra that, when compared to spectra obtained from protein-free bilayers, indicated that lipid chain motions are restricted by interactions with the protein. From the fraction of the motionally restricted component obtained from the phosphatidylcholine spin-label, a value of 64 +/- 14 annular lipids per band 3 dimer was obtained. This agrees with a value of 62 for the number of lipids that may be accommodated around the electron density map of a band 3 dimer. Selectivity of various spin-labeled lipids for the protein revealed that androstanol had a lower affinity for the band 3 interface, whereas a distinct preference was observed for the negatively charged lipids phosphatidylglycerol and stearic acid over phosphatidylcholine. This preference for negatively charged lipids could not be screened by 1-M salt, indicating that electrostatic lipid-protein interactions are not dominant. Estimates of annular lipid exchange rates from measured acyl chain segmental motions suggested that the rate of exchange between bilayer and boundary lipids was approximately 10(6) s(-1), at least an order of magnitude slower than the rate of lipid lateral diffusion in protein-free bilayers.     

Incorporation of the V-ATPase inhibitors concanamycin and indole pentadiene in lipid membranes. Spin-label EPR studies

The incorporation of concanamycin A, a potent inhibitor of vacuolar ATPases, into membranes of dimyristoyl phosphatidylcholine has been studied by using EPR of spin-labelled lipid chains. At an inhibitor/lipid ratio of 1:1 mol/mol, concanamycin A broadens the chain-melting transition of the phospholipid bilayer membrane, and effects the lipid chain motion in the fluid phase. The outer hyperfine splitting of a spin label at the C-5 position and the line widths of a spin label at the C-14 position of the lipid chain are increased by concanamycin A. Considerably larger membrane perturbations are caused by equimolar admixture of a designed synthetic 5-(5,6-dichloro-2-indolyl)-2,4-pentadienoyl V-ATPase inhibitor. These results indicate that concanamycin A intercalates readily between the lipid chains in biological membranes, with minimal perturbation of the bilayer structure. Essentially identical results are obtained with concanamycin A added to preformed membranes as a concentrated solution in DMSO, or mixed with lipid in organic solvent prior to membrane formation. Therefore, the common mode of addition in V-ATPase inhibition assays ensures incorporation of concanamycin into the lipid bilayer milieu, which provides an efficient channel of access to the transmembrane domains of the V-ATPase.     

Spin-label electron spin resonance studies of micellar dispersions of PEGs-PEs polymer-lipids

Conventional electron spin resonance (ESR) spectroscopy of different positional isomers of phosphatidylcholine spin labels (n-PCSL; n=5, 7, 10, 12, 14, and 16) has been used to study micellar dispersions made of poly(ethylene glycol)s-phosphatidylethanolamines (PEGs-PEs) polymer-lipids. Such aggregates are currently used as long circulating drug delivery systems "in vivo." We varied both the hydrocarbon chain length and the polymer size of the polymer-lipids. The dependence of the lipid-chain packing density on temperature and on label position as well as the flexibility and polarity profiles with position of chain labeling have been established for the PEGs-PEs micellar dispersions. The results show both similarity and differences either with common micellar dispersions of single chained lyso-palmitoylphosphatidylcholine (C(16)Lyso-PC) or with lamellar dispersions of double chained dipalmitoylphosphatidylcholine (DPPC). Well defined chain flexibility gradients of the same overall shape are obtained in the considered dispersions. However, the mobility of the first acyl chain segments is appreciable higher in micelles of polymer-lipids than in bilayers of DPPC and it becomes indistinguishable at the chain termini. A trend of decreasing polarity on moving toward the bilayer interior is seen in DPPC bilayers, whereas biphasic polarity profiles are obtained in micelles of polymer-lipids and C(16)Lyso-PC. Moreover, the properties of the PEGs-PEs micelles do not depend on the length of the hydrocarbon chain of the polymer-lipids but are slightly influenced by the size of the polymer.