Spin label saturation transfer ESR studies of protein-lipid interactions in Photosystem II-enriched membranes

Saturation transfer ESR has been used to study the dynamic behaviour of lipids in the appressed regions of thylakoid membranes from pea seedlings. Four different phospho- and galacto-lipid spin labels (phosphatidylcholine labelled at the 12 or 14 C-atom positions of the sn-2 chain, phosphatidylglycerol labelled at the 14-position of the sn-2 chain, and monogalactosyldiacylglycerol labelled at the 12-position of the sn-2 chain) were used to probe the lipid environment in photosystem II-enriched membranes prepared by detergent extraction. The ESR spectra show that the majority of the lipid in these preparations is strongly motionally restricted. Values for the effective rotational correlation times of the labelled chains were deduced from the lineheight ratios and integrals of thhe saturation transfer ESR spectra. The effective rotational correlation times were found to be in the 10⁵ range, indicating a very low lipid chain mobility which correlates with the low lipid content of these preparations. Comparison of the effective rotational correlation times deduced from the different diagnostic regions of the spectrum revealed little anisotropy in the chain mobility, indicating that the dominant motional mode was trans-gauche isomerization. The effective rotational correlation times deduced from the spectral integrals were similar to those deduced from the lineheight ratios, consistent with the absence of any appreciable fluid lipid component in these preparations. The results also indicate some selectivity of interaction between the lipid species, with phosphatidylcholine exhibiting appreciably slower motion than either phosphatidylglycerol or monogalactosyldiacylglycerol.     

Cooperative lipid activation of (Na+ + K+)-ATPase as a consequence of non-cooperative lipid-protein interactions

Lipid activation data for $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (Ottolenghi, P. (1979) Eur. J. Biochem. 99, 113–131) have been subjected to a regression and fitting analysis based on a recent kinetic model (Sandermann, H. (1982) Eur. J. Biochem, 127, 123–128). The observed kinetic cooperativity could be generated from strictly non-cooperative binding events involving the known number of 30 boundary lipid-binding sites per ATPase monomer. Apparent lipid dissociation equilibrium constants of between 0.3 and 5 μM were obtained, enzyme activity being associated only with the fully lipid-substituted enzyme and enzyme-lipid complexes with less than six unoccupied lipid-binding sites. The enzyme appeared to operate close to a maximum of cooperativity.     

Distinct states of lipid mobility in bovine rod outer segment membranes Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0°C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24°C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0°C and a considerable temperature dependence. This component which is not resolved at high temperatures (24–35°C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62–64 G at 0°C, with very little temperature dependence (61–62 G at 35°C), and is attributed to protein aggregation.     

Lipid dependence of peptide-membrane interactions: Bilayer affinity and aggregation of the peptide alamethicin

Membrane incorporation and aggregation of the peptide alamethicin have been investigated as a function of lipid type. Head group and acyl chain regions both contribute to modulate alamethicin incorporation. Specifically, the peptide prefers thin membranes and saturated chains; incorporation is reduced by the presence of cholesterol. Aggregation of the peptide in the bilayer is virtually insensitive to changes in lipid composition. These findings show some analogies to results obtained with intrinsic membrane proteins and cast doubt on the use of global membrane parameters for interpreting lipid-peptide interactions.     

Pressure effects on lipid-protein interactions in (Na+ + K+)-ATPase

The (Na⁺ + K⁺)-stimulated ATPase activity decreases with increasing pressure and a plot of the logarithm of the activity versus pressure shows a change in slope at a defined breakpoint pressure (P_b). The value of P_b increases linearly with increasing temperature. A $\text{d}\text{T}\text{d}\text{P}$ value of 27.7 ± 0.4 (S.D.) K/1000 atm is obtained. This is in very good agreement with the pressure shift for the melting transitions in phospholipids and aliphatic chains. This strongly indicates that an aliphatic chain melting process is involved in the breakpoint in the Arrhenius plot and pressure dependence of (Na⁺ + K⁺)-ATPase. The p-nitrophenyl phosphatase activity of this enzyme also decreases with pressure. In this case the plot of the logarithm of the activity versus pressure is linear without a break-point. The temperature dependence for (Na⁺ + K⁺)-ATPase was also studied in the presence of fluidizing drugs: desipramine and benzylalcohol. The presence of these drugs had no effect on the inflection point in the Arrhenius plot.     

温石棉对人红细胞膜脂质及蛋白质影响的ESR研究

用５ＮＳ、１６ＮＳ及ＭＳＬ标记人红细胞膜,观察了茫崖及涞温源石棉对膜脂质及蛋白质的影响。结果表明,两种温石棉均可增加膜表面层及深层脂质刚性,即致膜表、深层脂质流动性降低;同时可改变膜蛋白构象。两种温石棉时膜脂质及蛋白质的影响强度与其剂量有关。经柠檬酸铝处理后,温石棉的上述作用明显减弱。     

A spin label study of the urinary bladder luminal membrane

Spin label experiments have been carried out on the urinary bladder luminal membrane of the bovine transitional epithelium employing the 5-, 7-, 12-, and 16-doxyl substituted stearic acid methyl esters, and compared for reference to similarly labeled bovine erythrocytes. The bladder membranes are significantly different from the bovine red blood cell membranes and show a lower order and polarity near the membrane surface. This fact and the general similarity of results for the bladder and isolated plaque membranes suggests that the highly organized proteins of the bladder membrane may act as a coat on the lipid bilayer and, while intrinsic in nature, do not significantly perturb the hydrophobic core of the lipid bilayer.     

Properties of the strongly immobilized signal observed in spin-labeled erythrocytes

A strongly immobilized signal from fatty acid spin labels was observed in human erythrocytes treated with oxidizing agents such as glutaraldehyde, hydrogen peroxide, phenylhydrazine and copper-ortho-phenanthroline. This signal was also observed in freshly prepared ghosts treated with potassium superoxide and in old erythrocyte ghosts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these samples demonstrated the diffuse, nondiscrete bands of high molecular weight due to the cross-linking of membrane proteins. The temperature and pH dependences of the outer hyperfine splitting of this signal were very similar to those of bovine serum albumin. We propose that the strongly immobilized signal reflects the interaction of the lipids with the cross-linked products of membrane proteins.     

ESR spectral changes induced by chlorpromazine in spin-labeled erythrocyte ghost membranes

chlorpromazine interacted preferentially with membrane proteins rather than membrane lipids in the initial incorporation into human erythrocyte ghosts, as demonstrated by means of the fluorescence quenching and a maleimide spin label. In this state the membrane fluidity increased. At higher concentrations of chlorpromazine, the membrane fluidity decreased and a motionally restricted signal from fatty acid spin labels appeared predominantly. However, no such signal appeared in protein-free vesicles. The temperature and pH dependences of the outer hyperfine splitting of this restricted signal were very similar to those of bovine serum albumin. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorpromazine-treated and -untreated ghosts, it was found that there was no significant difference in membrane proteins between both samples except for the changes of a few bands which were not directly concerned with the occurrence of this restricted signal. These results suggest that the fatty acid spin labels bind preferably to membrane proteins as the lipid domain becomes packed with chlorpromazine.     

ESR Spectroscopy Investigation of the Denaturation Process of Soybean Peroxidase Induced by Guanidine Hydrochloride, DMSO or Heat

The involvement of protein denaturation and/or misfolding processes in the insurgence of several diseases raises the interest in structural dynamic studies of proteins. The use of nitroxide spin labels with electron paramagnetic resonance is a powerful tool for detecting structural changes in proteins. In the present study, we apply this strategy to soybean peroxidase (SBP), a protein characterised by high thermal and structural stability, and we propose a simple method to analyse the anisotropy changes of the protein system and to relate them with the structural changes induced by protein unfolding. We examined the effect of temperature, guanidine hydrochloride and dimethylsulfoxide on the stability of SBP and looked for correlations between the ESR results and the experimental findings obtained by other techniques, reported in the literature. The agreement between data obtained through different strategies supports the validity and reliability of the ESR approach to protein unfolding.     

Interaction of human serum albumin with membranes containing polymer-grafted lipids: spin-label ESR studies in the mushroom and brush regimes

The adsorption of human serum albumin (HSA) to dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was studied as a function of content and headgroup size of the polymer lipid. In the absence of protein, conversion from the low-density mushroom regime to the high-density brush regime of polymer-lipid content is detected by the change in ESR outer hyperfine splitting, 2A_max, of chain spin-labelled phosphatidylcholine in gel-phase membranes. The values of 2A_max remain constant in the mushroom regime, but decrease on entering the brush regime. Conversion between the two regimes occurs at mole fractions X_PEG(m→b)≈0.04, 0.01-0.02 and 0.005-0.01 for PEG-DPPE with mean PEG molecular masses of 350, 2000 and 5000 Da, respectively, as expected theoretically. Adsorption of HSA to DPPC membranes is detected as a decrease of the spin label 2A_max hyperfine splitting in the gel phase. Saturation is obtained at a protein/lipid ratio of ca. 1:1 w/w. In the presence of polymer-grafted lipids, HSA adsorbs to DPPC membranes only in the mushroom regime, irrespective of polymer length. In the brush regime, the spin-label values of 2A_max are unchanged in the presence of protein. Even in the mushroom regime, protein adsorption progressively becomes strongly attenuated as a result of the steric stabilization exerted by the polymer lipid. These results are in agreement with theoretical estimates of the lateral pressure exerted by the grafted polymer in the brush and mushroom regimes, respectively.     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes: A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This ‘stiffening’ effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35°C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This corresponds to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10°C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Lipid-protein interactions in biological membranes: a structural perspective

Lipid molecules bound to membrane proteins are resolved in some high-resolution structures of membrane proteins. An analysis of these structures provides a framework within which to analyse the nature of lipid-protein interactions within membranes. Membrane proteins are surrounded by a shell or annulus of lipid molecules, equivalent to the solvent layer surrounding a water-soluble protein. The lipid bilayer extends right up to the membrane protein, with a uniform thickness around the protein. The surface of a membrane protein contains many shallow grooves and protrusions to which the fatty acyl chains of the surrounding lipids conform to provide tight packing into the membrane. An individual lipid molecule will remain in the annular shell around a protein for only a short period of time. Binding to the annular shell shows relatively little structural specificity. As well as the annular lipid, there is evidence for other lipid molecules bound between the transmembrane α-helices of the protein; these lipids are referred to as non-annular lipids. The average thickness of the hydrophobic domain of a membrane protein is about 29 Å, with a few proteins having significantly smaller or greater thicknesses than the average. Hydrophobic mismatch between a membrane protein and the surrounding lipid bilayer generally leads to only small changes in membrane thickness. Possible adaptations in the protein to minimise mismatch include tilting of the helices and rotation of side chains at the ends of the helices. Packing of transmembrane α-helices is dependent on the chain length of the surrounding phospholipids. The function of membrane proteins is dependent on the thickness of the surrounding lipid bilayer, sometimes on the presence of specific, usually anionic, phospholipids, and sometimes on the phase of the phospholipid.     

Incorporation of the antimicrobial protein seminalplasmin into lipid bilayer membranes

The interaction between seminalplasmin, an antimicrobial protein from bull semen, and lipid bilayers has been investigated. The fluorescence of the single tryptophan residue of the protein was measured. In the presence of phosphatidylcholine or phosphatidic acid bilayer vesicles the fluoresence maximum was shifted to shorter wavelengths, indicating transfer of the tryptophan to a more apolar environment. Circular dichroism spectra show an increased -helical content for the protein in the presence of lipid. Quenching experiments clearly show the incorporation of the protein with the tryptophan localized near the bilayer surface. The shift of the tryptophan fluorescence emission was used to monitor the lipid phase transition in phosphatidylcholine membranes.Abbreviations TEMPOL 2,2,6,6-Tetramethyl-4-hydroxy-piperidine-1-oxyl - DMPC 1,2-Dimyristoylphosphatidylcholine - DMPA 1,2-Dimyristoylphosphatidic acid - SL 5 2-(3-Carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinoxyl - SL 12 2-(10-Carboxydecyl)-4,4-dimethyl-2-hexyl-3-oxazolinoxyl     

Lipid-protein interactions: NMR study of melittin and its binding to lysophosphatidylcholine

Proton NMR of melittin differs according to the association state of the peptide in the monomer or tetramer. Melittin interacts with lysophosphatidylcholine micelles, whatever the association state of melittin; well resolved superimposed spectra from both components for all the lipid to peptide molar ratios are observed. Within the complexes, local mobility and fast exchange occurs. On binding concomitant shifts on Trp₁₉ indole lines and on the aliphatic CH₂ protons of the lipids are detected. The lipid perturbation is maximum for methylene groups in α and β of the ester bond, this could allow positionning of Trp₁₉ in the hydrophobic core of the lipids.     

Phosphatidylcholine and cholesterol interactions in model membranes

Various phosphatidylcholines differing either in the stereochemistry around their chiral center or in the position of a cis double bond along the acyl chains were synthesized in order to study critical contact regions in the phospholipid molecule with adjacent cholesterol in model membranes. Microviscosities calculated from fluorescence depolarization of diphenylhexatriene and chain order from spin label studies were measured to monitor physical membrane properties. The enhancing effect of cholesterol on the microviscosity of membranes containing phosphatidylcholines with comparable acyl chain length was largest when the two acyl chains were saturated and smallest when both were unsaturated. Membranes prepared from phosphatidylcholines having a single cis double bond at different positions along the sn-2 acyl chain showed roughly the same changes of microviscosity or chain order upon incorporation of cholesterol. No discrimination was evident in the interaction between cholesterol and enantiomeric phosphatidylcholines or between the enantiomeric phosphatidylcholine molecules themselves. We conclude that the rigidifying effect of cholesterol in membranes does not depend on specific sites of interaction and that with respect to physical membrane properties phosphatidylcholine behaves as an achiral molecule.     

The role of chromophore in the lipid-protein interactions in bacteriorhodopsin-phosphatidylcholine vesicles

By fluorescence and phase properties of a 1-acyl-2-[8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine probe, the influence of the chromophore on the phase transition of bacteriorhodopsin–lipid vesicles was investigated. It was observed that removal of the chromophore led to the down-shifting of the phase transition temperatures. The temperatures corresponding to the beginning and ending of the gel–liquid phase transition were also influenced. This demonstrated that the liquid phase is reached more easily when the chromophore is bleached. The results indicate that removal of the chromophore alters the protein–lipid interactions. It is suggested that this alteration might be related to the change in the lipid molecular packing.     

Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes: Lipid specificity and effect of the temperature

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins. The affinity of the protein BSP-A1/-A2 for lipid membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and POPC containing 30% (mol/mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or cholesterol, has been investigated by the isothermal titration calorimetry (ITC). This study confirms the association of these proteins to lipid bilayers, and provides a direct characterization of this exothermic process, at 37 °C. The measurements indicate that the protein affinity for lipid bilayers is modulated by the lipid composition, the lipid/protein ratio, and the temperature. The saturation lipid/protein ratio was increased in the presence of cholesterol and, to a lesser extent, of phosphatidylethanolamine, suggesting that it is modulated by the lipid acyl chain order. For all the investigated systems, the binding of BSP-A1/-A2 could not be modeled using a simple partitioning of the proteins between the aqueous and lipid phases. The existence of "binding sites", and lipid phase separations is discussed. The decrease of temperature, from 37 to 10 °C, converts the exothermic association of the proteins to the POPC bilayers to an endothermic process. A complementary 1-D and 2-D infrared spectroscopy study excludes the thermal denaturation of BSP-A1/-A2 as a contributor in the temperature dependence of the protein affinity for lipid bilayers. The reported findings suggest that changes in the affinity of BSP-A1/-A2 for lipid bilayers could be involved in modulating the association of these proteins to sperm membranes as a function of space and time; this would consequently modulate the extent of lipid extraction, including cholesterol, at a given place and given time.     

Interaction of 3-Alkylpyridinium Polymers from the Sea Sponge Reniera sarai with Insect Acetylcholinesterase

3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS–PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS–PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.     

The adsorption of adrenocorticotropin-(1-24)-tetracosapeptide to lecithin bilayer membranes formed from liposomes

The interaction of the peptide hormone adrenocorticotropin (ACTH_1-24) with solvent-free planar lipid bilayers has been studied by use of the capacitance minimization method. The membranes were formed from artificial vesicles according to the method described by Schindler. In contrast to analogous studies with hexane-containing membranes, experiments with these vesicle-derived bilayers were completely reproducible and gave no indication that ACTH_1-24 spans such hexane-free bilayers.