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PubMed1989

Selectivity of interaction of phospholipids with bovine spinal cord myelin basic protein studied by spin-label electron spin resonance.

The selectivity of interaction between bovine spinal cord myelin basic protein (MBP) and eight different spin-labeled lipid species in complexes with dimyristoylphosphatidylglycerol (DMPG) and between spin-labeled phosphatidylglycerol and spin-labeled phosphatidylcholine in complexes of MBP with various mixtures of DMPG and dimyristoylphosphatidylcholine (DMPC) has been studied by electron spin resonance (ESR) spectroscopy. In DMPC/DMPG mixtures, the protein binding gradually decreased with increasing mole fraction of DMPC in a nonlinear fashion. The lipid-protein binding assays indicated a preferential binding of the protein to phosphatidylglycerol relative to phosphatidylcholine without complete phase separation of the two lipids. The outer hyperfine splittings (2Amax) of both phosphatidylglycerol and phosphatidylcholine labeled at C-5 of the sn-2 chain (5-PGSL and 5-PCSL, respectively) were monitored in the lipid-protein complexes as a function of the mole fraction of DMPC. The increases in the value of Amax induced on binding of the protein were larger for 5-PGSL than for 5-PCSL, up to 0.25 mole fraction of DMPC. Beyond this mole fraction the spectral perturbations induced by the protein were similar for both lipid labels. The ESR spectra of phosphatidylglycerol and phosphatidylcholine labeled at C-12 of the sn-2 chain were two component in nature, indicating indicating a direct interaction of the protein with the lipid chains, at mole fractions of DMPC up to 0.25. Quantitation of the motionally restricted spin-label population by spectral subtraction again indicated a preferential interaction of the protein with phosphatidylglycerol relative to phosphatidylcholine. Up to DMPC mode fractions of 0.25, the microenvironment of the protein was enriched in DMPG.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

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.

Drug Stability↗
PubMed1986

Apocytochrome c binding to negatively charged lipid dispersions studied by spin-label electron spin resonance.

The interaction of apocytochrome c with aqueous dispersions of phosphatidylserine from bovine spinal cord and with other negatively charged phospholipids has been studied as a function of pH and salt concentration by using spin-label electron spin resonance (ESR) spectroscopy and chemical binding assays. The ESR spectra of phospholipids spin-labeled at different positions on the sn-2 chain indicate a generalized decrease in mobility of the lipids, while the characteristic flexibility gradient toward the terminal methyl end of the chain is maintained, on binding of apocytochrome c to phosphatidylserine dispersions. This perturbation of the bulk lipid mobility or ordering is considerably greater than that observed on binding of cytochrome c. In addition, a second, more motionally restricted, lipid component is observed with lipids labeled close to the terminal methyl ends of the chains. This second component is not observed on binding of cytochrome c and can be taken as direct evidence for penetration of apocytochrome c into the lipid bilayer. It is less strongly motionally restricted than similar spectral components observed with integral membrane proteins and displays a steep flexibility gradient. The proportion of this second component increases with increasing protein-to-lipid ratio, but the stoichiometry per protein bound decreases from 4.5 lipids per 12 000-dalton protein at low protein contents to 2 lipids per protein at saturating amounts of protein. Apocytochrome c binding to phosphatidylserine dispersions decreases with increasing salt concentration from a saturation value corresponding to approximately 5 lipids per protein in the absence of salt to practically zero at 0.4 M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Lipid-protein interactions in thylakoid membranes of chilling-resistant and -sensitive plants studied by spin label electron spin resonance spectroscopy.

Lipid-protein interactions in thylakoid membranes from lettuce, pea, tomato, and cucumber have been studied using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyl diglyceride and phosphatidylglycerol. 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 lipids interacting with the integral membrane proteins. Comparison of the spectra from the same spin label in thylakoid membranes from different plants shows that the overall lipid fluidity in the membranes decreases with chilling sensitivity. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with integral membrane proteins. Thylakoid membranes of cucumber, a typical chilling-sensitive plant, have been found to have a higher proportion of motionally restricted lipids and a different lipid selectivity for lipid-protein interaction, as compared with those of pea, a typical chilling-resistant plant. This correlation with chilling sensitivity holds generally for the different plants studied. It seems likely that the chilling sensitivity in thylakoid membranes is not determined by lipid fluidity alone, but also by the lipid-protein interactions which could affect protein function in a more direct manner.

Cold Temperature↗
PubMed1989

Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study.

The contribution of the various regions of the mitochondrial precursor protein apocytochrome c to the interaction of the protein with phosphatidylserine dispersions has been studied with chemically and enzymatically prepared fragments of horse heart apocytochrome c and phospholipids spin-labeled at different positions of the sn-2 chain. Three amino-terminal heme-less peptides, two heme-containing amino-terminal fragments, one central fragment, and three carboxy-terminal fragments were studied. The electron spin resonance spectra of phospholipids spin-labeled at the C5 position of the fatty acid chain indicate that both amino-terminal and carboxy-terminal fragments of the apocytochrome c molecule cause a restriction of motion of the lipids, whereas the heme-containing peptides and protein have less effect. In addition, a second motionally more restricted lipid component, which is observed for apocytochrome c interacting with phosphatidylserine dispersions containing lipids spin-labeled at the C12 or C14 position [Görrissen, H., Marsh, D., Rietveld, A., & de Kruijff, B. (1986) Biochemistry 25, 2904-2910], was observed both on binding the carboxy-terminal fragments and on binding of the amino-terminal fragments of the precursor protein. Interestingly, even a small water-soluble peptide consisting of the 24 carboxy-terminal residues gave rise to a two-component spectrum, with an outer hyperfine splitting of the restricted lipid component of 59 G, indicating a considerable restriction of the chain motion. This suggests that both the carboxy- and amino-terminal parts of the protein penetrate into the center of the bilayer and cause a strong perturbation of the fatty acyl chain motion. The implications of these findings for the mechanism of apocytochrome c translocation across membranes are discussed.

Animals↗
PubMed2005

Domain formation in sphingomyelin/cholesterol mixed membranes studied by spin-label electron spin resonance spectroscopy.

Interactions of palmitoylsphingomyelin with cholesterol in multilamellar vesicles have been studied over a wide range of compositions and temperatures in excess water by using electron spin resonance (ESR) spectroscopy. Spin labels bearing the nitroxide free radical group on the 5 or 14 C-atom in either the sn-2 stearoyl chain of phosphatidylcholine (predominantly 1-palmitoyl) or the N-stearoyl chain of sphingomyelin were used to determine the mobility and ordering of the lipids in the different phases. Two-component ESR spectra of the 14-position spin labels demonstrate the coexistence first of gel (L(beta)) and liquid-ordered (L(o)) phases and then of liquid-ordered and liquid-disordered (L(alpha)) phases, with progressively increasing temperature. These phase coexistences are detected over a limited range of cholesterol contents. ESR spectra of the 5-position spin labels register an abrupt increase in ordering at the L(alpha)-L(o) transition and a biphasic response at the L(beta)-L(o) transition. Differences in outer splitting between the C14-labeled sphingomyelin and phosphatidylcholine probes are attributed to partial interdigitation of the sphingomyelin N-acyl chains across the bilayer plane in the L(o) state. In the region where the two fluid phases, L(alpha) and L(o), coexist, the rate at which lipids exchange between phases (<<7 x 10(7) s(-)(1)) is much slower than translational rates in the L(alpha) phase, which facilitates resolution of two-component spectra.

Cholesterol↗
PubMed1988

Exchange rates at the lipid-protein interface of myelin proteolipid protein studied by spin-label electron spin resonance.

The electron spin resonance (ESR) spectra from spin-labeled phospholipids in recombinants of myelin proteolipid apoprotein with dimyristoylphosphatidylcholine have been simulated with the exchanged-coupled Bloch equations to obtain values for both the fraction of motionally restricted lipids and the exchange rate between the fluid and motionally restricted lipid populations. The rate of exchange between the two spin-labeled lipid components is found to lie in the slow exchange regime of nitroxide ESR spectroscopy. The values obtained for the fraction of motionally restricted component in the exchanged-coupled spectra are found to be in good agreement with those obtained previously by spectral subtraction for the same system [Brophy, P. J., Horváth, L. I., & Marsh, D. (1984) Biochemistry 23, 860-865]. The rate of lipid exchange off the protein is independent of lipid/protein ratio for a given spin-labeled phospholipid, as expected, and decreases with increasing selectivity of the various phospholipids for the protein. At 30 degrees C and for ionic strength 0.1 and pH 7.4, the off-rate constants are 4.6 X 10(6) s-1 for phosphatidic acid, 1.1 X 10(7) s-1 for phosphatidylserine, 1.6 X 10(7) s-1 for phosphatidylcholine, and 2.2 X 10(7) s-1 for phosphatidylethanolamine. These values are in the inverse ratio of the relative association constants of the various lipids for the protein (Brophy et al., 1984) and are appreciably slower than the rate of lipid lateral diffusion in dimyristoylphosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Dimyristoylphosphatidylcholine↗
PubMed1999

Membrane lysis by the antibacterial peptides cecropins B1 and B3: A spin-label electron spin resonance study on phospholipid bilayers.

Custom antibacterial peptides, cecropins B1 (CB1) and B3 (CB3), were synthesized. These peptides have particular sequence characteristics, with CB1 having two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of their efficacy in breaking up liposomes, which had different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their lipid binding ability. Binding and nonbinding lysis actions of CB1 and CB3 on liposomes were examined further by electron spin resonance (ESR). The spin-labeled lipids 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC, and 16'SL-PC were used as probes. The ESR spectra revealed larger outer hyperfine splittings (2A(max)) for CB1 when the interactions of CB1 and CB3 with liposomes were compared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of CB1. Plots of the effective order parameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. To augment these findings, four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3, and C30SL-CB3, were also examined for their binding to and their state of aggregation within the lipid bilayers. Association isotherms of the peptides were measured for liposomes containing two molar fractions of PA (0.25 and 0.75). The membrane binding of the CB1 peptides exhibited a cooperative behavior, whereas the association isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposomes. To further identify the location of CB1 in the lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggested that the NH(2)-terminal alpha-helix of CB1 is located on the surface of the lipid bilayers, whereas the COOH-terminal alpha-helix of CB1 is embedded below the surface of the lipid bilayers. These conclusions were further supported by the observed relationship between the partition distribution of peptides bound to liposomes at different PA/PC ratios and the amounts of free peptides. Based on the above observations, possible mechanisms of the bilayer lysis induced by CB1 and CB3 on liposomes of different composition are discussed.

Amino Acid Sequence↗
PubMed2004

Transfer of stearic acids from albumin to polymer-grafted lipid containing membranes probed by spin-label electron spin resonance.

Human serum albumin (HSA) has been spin-labelled with stearic acids having the nitroxide moiety attached to the hydrocarbon chain either at the 5th or at the 16th carbon atom (n-SASL, n = 5 and 16, respectively) with respect to the carboxyl groups. Its interaction with sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) mixed with submicellar content of poly(ethylene glycol:2000)-grafted dipalmitoyl phosphatidylethanolamine (PEG:2000-DPPE) has been studied by conventional electron spin resonance (ESR) spectroscopy. In the absence of bilayer membranes, the ESR spectra of nitroxide stearic acids non-covalently bound to HSA are single component powder patterns, indicative of spin labels undergoing temperature dependent anisotropic motion in the slow motional regime on the conventional ESR timescale. The adsorption of HSA to DPPC bilayers results in two component ESR spectra. Indeed, superimposed to an anisotropic protein-signal appears a more isotropic signal due to the labels in the lipid environment. This accounts for the transfer of fatty acids from the protein to DPPC bilayers. Two spectral components with different rotational mobility are also singled out in the spectra of n-SASL bound to HSA when DPPC/PEG:2000-DPPE mixtures are present in the dispersion medium. The fraction, f(L)(16-SASL), of spin labels transferred from the protein to lipid/polymer-lipid lamellar membranes has been quantified performing spectral subtraction. It is found that f(L)(16-SASL) decreases on increasing the content of the polymer-lipid mixed with DPPC. It is strongly reduced in the low-density mushroom regime and levels off in the high-density brush regime of the polymer-lipid content as a result of the steric stabilization exerted by the PEG-lipids. Moreover, the fraction of transferred fatty acids from HSA to SSL is dependent on the physical state of the lipid bilayers. It progressively increases with increasing the temperature from the gel to the liquid-crystalline lamellar phases of the mixed lipid/polymer-lipid membranes, although such a dependence is much weaker in the brush regime.

Electron Spin Resonance Spectroscopy↗
PubMed1995

Lipid-protein interactions and assembly of the 16-kDa channel polypeptide from Nephrops norvegicus. Studies with spin-label electron spin resonance spectroscopy and electron microscopy.

The assembly of 16-kDa polypeptide channel units in membranes from the hepatopancreas of Nephrops norvegicus has been studied both by electron microscopy and by the lipid--protein interactions reported with spin-labeled lipids. Membranes prepared by extraction with N-lauroylsarcosine and Triton X-100 have a low lipid/protein ratio (ca. 4-6.5 phospholipids and 1 cholesterol per 16-kDa monomer), and those prepared by alkaline extraction have a higher lipid/protein ratio (ca. 12-16 phospholipids and 3.5-4 cholesterols per 16-kDa monomer). In the membranes extracted with detergents, the protein is assembled in membrane sheets as hexagonally packed hexameric complexes, whereas the alkali-extracted preparations consist of closed vesicles in which the channel complexes are near randomly distributed. The electron spin resonance (ESR) spectra from lipids spin-labeled at the C-14 position of the (sn-2) chain show lower mobility for the membranes extracted with N-lauroylsarcosine than for the alkaline-extracted membranes. At higher temperatures, the ESR spectra reveal a population of lipids whose mobility is restricted by direct interaction with the intramembranous sections of the channel assemblies. The population of protein-associated spin-labeled phosphatidylcholine in the alkali-extracted membranes corresponds to 4-5 phospholipid molecules plus 1 cholesterol molecule per 16-kDa polypeptide monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Interaction of bee venom melittin with zwitterionic and negatively charged phospholipid bilayers: a spin-label electron spin resonance study.

Electron spin resonance (ESR) spectroscopy was used to study the penetration and interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) and ditetradecylphosphatidylglycerol (DTPG) bilayer membranes. Melittin is a surface-active, amphipathic peptide and serves as a useful model for a variety of membrane interactions, including those of presequences and signal peptides, as well as the charged subdomain of the cardiac regulatory protein phospholamban. Derivatives of phosphatidylcholine and phosphatidylglycerol spin-labeled at various positions along the sn-2 acyl chain were used to establish the chain flexibility gradient for the two membranes in the presence and absence of melittin. Negatively charged DTPG bilayer membranes showed a higher capacity for binding melittin without bilayer disruption than did membranes formed by the zwitterionic DMPC, demonstrating the electrostatic neutralization of bound melittin by DTPG. The temperature dependence of the ESR spectra showed that the gel-to-liquid crystalline phase transition is eliminated by binding melittin to DTPG bilayers, whereas a very broad transition remains in the case of DMPC bilayers. None of the spin labels used showed a two-component spectrum characteristic of a specific restriction of their chain motion by melittin, but the outer hyperfine splittings and effective chain order parameters were increased for all labels upon binding melittin. This indicates a reduced flexibility of the lipid chains induced by a surface orientation of the bound melittin. Whereas the characteristic shape of the chain flexibility gradient was maintained upon melittin addition to DMPC bilayers, the chain flexibility profile in DTPG bilayers was much more strongly perturbed. It was found that the steepest change in segmental flexibility was shifted toward the bilayer interior when melittin was bound to DTPG membranes, indicating a greater depth of penetration than in DMPC membranes. pH titration of stearic acid labeled at the C-5 position, used as a probe of interfacial interactions, showed net downward shifts in interfacial pK of 0.8 and 1.2 pH units contributed from the positive charge of melittin, outweighing upward shifts from interfacial dehydration, when melittin was bound to DTPG and DMPC, respectively. The perturbation of the outer hyperfine splitting was used to determine the interactions of melittin with spin-labeled lipids of different polar headgroups in DTPG and DMPC. Anionic lipids (phosphatidylserine, phosphatidylglycerol, and stearic acid) and zwitterionic lipids (phosphatidylethanolamine and phosphatidylcholine) had the largest outer splittings in the presence of melittin. Neutral lipids (protonated stearic acid and diacylglycerol) displayed the largest increase in outer splitting on binding melittin, which was attributed to a change in the vertical location of these lipids in the bilayer. Both effects were more pronounced in DTPG than in DMPC.

Amino Acid Sequence↗
PubMed1992

Stoichiometry, selectivity, and exchange dynamics of lipid-protein interaction with bacteriophage M13 coat protein studied by spin label electron spin resonance. Effects of protein secondary structure.

Bacteriophage M13 major coat protein has been isolated with cholate and reconstituted in dimyristoyl- and dioleoylphosphatidylcholine (DMPC and DOPC, respectively) bilayers by dialysis. Fourier transform infrared spectra of DMPC/coat protein recombinants confirmed that, whereas the protein isolated by phenol extraction was predominantly in a beta-sheet conformation, the cholate-isolated coat protein contained a higher proportion of the alpha-helical conformation [cf. Spruijt, R. B., Wolfs, C. J. A. M., & Hemminga, M. A. (1989) Biochemistry 28, 9158-9165]. The cholate-isolated coat protein/lipid recombinants gave different electron spin resonance (ESR) spectral line shapes of incorporated lipid spin labels, as compared with those from recombinants with the phenol-extracted protein that were studied previously [Wolfs, C. J. A. M., Horváth, L. I., Marsh, D., Watts, A., & Hemminga, M. A. (1989) Biochemistry 28, 9995-10001]. Plots of the ratio of the fluid/motionally restricted components in the ESR spectra of spin-labeled phosphatidylglycerol were linear with respect to the lipid/protein ratio in the recombinants up to 20 mol/mol. The corresponding values of the relative association constants, Kr, and number of association sites, N1, on the protein were Kr approximately 1 and N1 approximately 4 for DMPC recombinants and Kr approximately 1 and N1 approximately 5 for DOPC recombinants. Simulation of the two-component lipid spin label ESR spectra with the exchange-coupled Bloch equations gave values for the off-rate of the lipids leaving the protein surface of 2.0 x 10(7) s-1 at 27 degrees C in DMPC recombinants and 3.0 x 10(7) s-1 at 24 degrees C in DOPC recombinants.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophages↗
PubMed1984

Stoichiometry and specificity of lipid-protein interaction with myelin proteolipid protein studied by spin-label electron spin resonance.

The interaction of spin-labeled lipids with the myelin proteolipid apoprotein in complexes with dimyristoylphosphatidylcholine of varying lipid/protein ratios has been studied with electron spin resonance spectroscopy. A first shell of approximately 10 lipids per 25 000-dalton protein is found to be motionally restricted by the protein interface. This stoichiometry is consistent with a hexameric arrangement of the protein in the membrane. A selectivity of the various spin-labeled lipids for the motionally restricted component at the protein interface is found in the order stearic acid greater than phosphatidic acid greater than cardiolipin approximately greater than phosphatidylserine greater than phosphatidylglycerol approximately equal to phosphatidylcholine greater than phosphatidylethanolamine greater than androstanol approximately greater than cholestane.

Animals↗
PubMed1987

Spin-label electron spin resonance study of bacteriophage M13 coat protein incorporation into mixed lipid bilayers.

The major coat protein of bacteriophage M13 was incorporated in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (80/20 w/w) vesicles probed with different spin-labeled phospholipids, labeled on the C-14 atom of the sn-2 chain. The specificity for a series of phospholipids was determined from a motionally restricted component seen in the electron spin resonance (ESR) spectra of vesicles with the coat protein incorporated. At 30 degrees C and pH 8, the fraction of motionally restricted phosphatidic acid spin-label is 0.36, 0.52, and 0.72 for lipid/protein ratios of 18, 14, and 9 mol/mol, respectively. The ESR spectra, analyzed by digital subtraction, resulted in a phospholipid preference following the pattern cardiolipin = phosphatidic acid greater than stearic acid = phosphatidylserine = phosphatidylglycerol greater than phosphatidylcholine = phosphatidylethanolamine. The specificities found are related to the composition of the target Escherichia coli cytoplasmic membrane.

Coliphages↗
PubMed1997

A single-residue deletion alters the lipid selectivity of a K+ channel-associated peptide in the beta-conformation: spin label electron spin resonance studies.

Lipid-peptide interactions with the 27-residue peptide of sequence KLEALYILMVLGFFGFFTLGIMLSYIR reconstituted as beta-sheet assemblies in dimyristoylphosphatidylcholine bilayers have been studied by electron spin resonance (ESR) spectroscopy with spin-labeled lipids. The peptide corresponds to residues 42-68 of the IsK voltage-gated K+ channel protein and contains the single putative transmembrane span of this protein. Lipid-peptide interactions give rise to a second component in the ESR spectra of lipids spin-labeled on the 14C atom of the chain that corresponds to restriction of the lipid mobility by direct interaction with the peptide assemblies. From the dependence on the lipid/peptide ratio, the stoichiometry of lipid interaction is found to be about two phospholipids/peptide monomer. The sequence of selectivity for lipid association with the peptide assemblies is in the order phosphatidic acid > stearic acid = phosphatidylserine > phosphatidylglycerol = phosphatidylcholine. Comparison with previous data for a corresponding 26-residue mutant peptide with a single deletion of the apolar residue Leu2 (Horvath et al., 1995. Biochemistry 34:3893-3898), indicates a very similar mode of membrane incorporation for native and mutant peptides, but a strongly modified pattern and degree of specificity for the interaction with negatively charged lipids. The latter is interpreted in terms of the relative orientations of the charged amino acid side chains in the beta-sheet assemblies of the native and deletion-mutant peptides.

Amino Acid Sequence↗
PubMed2002

Immobilization-induced changes in erythrocyte membrane fluidity in rabbits: a spin-label electron spin resonance study.

It has been shown that many diseases are linked to abnormalities of the erythrocyte membrane. This study observed the changes in erythrocyte membrane fluidity during an immobilization period of 21 days. The right hindlimbs of male adult New Zealand white rabbits were immobilized for 21 days. Blood samples were collected and heparinized prior to immobilization and on days 1, 3, 7, 14, and 21 of immobilization. The membrane fluidity of erythrocytes was measured by spin-label electron spin resonance (ESR). The membrane fluidity was expressed by the value of order parameter (S). The results showed a significant increase of S on days 7 and 14 of immobilization (p <0.01). The highest value of S was found on day 7 of immobilization; thereafter, S gradually declined. Compared with the value measured before immobilization, there was no significant difference in the value of S on day 21. The results show that immobilization significantly affected erythrocyte membrane fluidity, indicating a systemic and temporal response ofthe immobilized body. This study is the first to document the time-course of changes in erythrocyte membrane fluidity in rabbits with single hindlimb immobilization during 21 days. These results may be helpful in studying the pathophysiology of immobilization.

Animals↗
PubMed1971

Spin-labeled electron spin resonance study of the lipid-containing membrane of influenza virus.

The organization of the lipid-containing membrane of influenza virus has been studied by the use of three different lipid spin labels, and the results are compared with a parallel study on human erythrocyte ghosts. The lipid phase of the viral membrane is slightly more rigid than that of the erythrocyte ghosts. The data suggest that the viral lipid is arranged in a bilayer. The data suggest that the viral lipid is arranged in a bilayer. The glycoprotein spikes covering the viral membrane were specifically removed by proteases, and no alteration in the environment of any of the three spin labels was detected. This suggests that the spikes are not involved in determining the organization of the lipid bilayer.

Cell Membrane↗
PubMed1982

Fluorescence quenching and spin label electron spin resonance studies of stacking self-association in aqueous solutions of 2-aminopurine riboside and its 5'-mono- and -diphosphate.

The autoassociation of 2-aminopurine riboside (rn2Pur) and its 5'-mono- (P-rn2Pur) and 5'-diphosphate (PP-rn2Pur) in neutral aqueous solutions was investigated using fluorescence quenching and ESR spin-label methods within the range 276-358 K. Respective equilibrium constants and thermodynamic functions were derived therefrom assuming two models of infinite autoassociation: (i) an isodesmic one (K2 = K3 = ... Kp), and (ii) one in which K2 no equal to K2 = K4 ... Kp. Comparative analysis of these data and that of the parent 2-aminopurine, obtained previously, allowed us to formulate the following conclusions: (1) the mechanism of autoassociation of rn2Pur varies with temperature in such a way that a T = 318 K the isodesmic model is fulfilled (K2 = Kp); at high temperatures Kp/K2 greater than 1, i.e. the process is cooperative, while at lower temperatures it becomes anticooperative (Kp/K2 greater less than 1); (2) at 298 K the tendency to autoassociation decreases in the order; rn2Pur greater than P-rn2Pur greater than PP-rn2Pur; (3) rn2Pur forms highly packed complexes with the bases stacked and the ribofuranose residues interacting via hydrogen bonds or water bridges; (4) autoassociation of P-rn2Pur and PP-rn2Pur is mainly governed by stacking of the bases, while the ribose phosphate residues attain a trans configuration corresponding to the lowest electrostatic repulsion between charged phosphate groups; even at high ionic strength (I = 0.8), a positive electrostatic contribution to the free enthalpy of autoassociation is observed; (5) the two methods employed gave similar results for P-rn2Pur, but somewhat different ones for rn2Pur because the presence of the spin label (nitroxide stable radical) at the 2'(3')-OH group of the ribose residues prevents its interaction via hydrogen bonding with an unlabeled one of an adjacent nucleoside.

Electron Spin Resonance Spectroscopy↗