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1.
Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from Squalus acanthias have been studied using spin-labeled derivatives of the mono- and disialogangliosides GM1, GM2, GM3, and GD1b, in conjunction with electron spin resonance (ESR) spectroscopy. Ganglioside-protein interactions are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component that corresponds closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled gangliosides whose chain motion is significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A small selectively for the motionally restricted component associated with the protein is found in the order GD1b greater than GM1 approximately equal to GM2 approximately equal to GM3. Comparison with previous results from spin-labeled phospholipids in the same system [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] shows that the spin-labeled monosialogangliosides GM1, GM2, and GM3 display little selectivity in the lipid-protein interaction relative to spin-labeled phosphatidylcholine. The spectral characteristics of both the fluid and motionally restricted spin-labeled components differ very significantly, however, between the gangliosides and the phospholipids. The outer hyperfine splitting of the motionally restricted component is smaller for the gangliosides than for the phospholipids, indicating a smaller degree of motional restriction on interaction of the ganglioside lipid chains with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

2.
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)  相似文献   

3.
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)  相似文献   

4.
5.
L I Horváth  P J Brophy  D Marsh 《Biochemistry》1988,27(14):5296-5304
The pH and salt dependences of the interaction of phosphatidic acid, phosphatidylserine, and stearic acid with myelin proteolipid apoprotein (PLP) in dimyristoylphosphatidylcholine (DMPC) recombinants have been studied by electron spin resonance spectroscopy, using spin-labeled lipids. The two-component spin-label spectra have been analyzed both by spectral subtraction and by simulation using the exchange-coupled Bloch equations to give the fraction of lipids motionally restricted by the protein and the rate of lipid exchange between the fluid and motionally restricted lipid populations. For stearic acid, phosphatidic acid, and phosphatidylserine, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 7.7, 7.6, and ca. 9.4, respectively. The corresponding pKa's for the bulk lipid regions of the bilayer are estimated, from changes in the ESR spectra, to be 6.7, 7.4, and 11, respectively. In the dissociated state at pH 9.0, the fraction of motionally restricted component decreases with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M for all three negatively charged lipids. The net decreases for stearic acid and phosphatidic acid are considerably smaller (by ca. 30%) than those obtained on protonating the two lipids, whereas for phosphatidylserine the fraction of motionally restricted lipid in high salt is reduced to that corresponding to phosphatidylcholine. For a fixed lipid/protein ratio, the on-rate for exchange at the lipid-protein interface is independent of the degree of selectivity and has a shallow temperature dependence, as expected for a diffusion-controlled process.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

6.
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)  相似文献   

7.
Stearic acids with a nitroxide radical at selected positions have been incorporated in the phospholipid bilayers of clathrin coated vesicles, uncoated vesicles and sonicated liposomes made from the lipids extracted from the uncoated vesicles. The extent of incorporation was found minimum for stearic acids labeled on C-12 and for bilayers of uncoated vesicles. The ESR spectra of the spin-labeled fatty acids incorporated in the bilayers showed a pronounced temperature dependence (without discontinuity) and a decrease in the hyperfine splitting as the nitroxide group was inserted deeper in the hydrophobic core of the membranes. An abrupt phospholipid phase transition or a phase separation could be excluded. The presence of the external proteins (the clathrin coat) on the membranes was not found to noticeably influence the gradient of flexibility of the fatty acid chains of the phospholipids. The influence of the internal proteins embedded in the bilayers was evidenced by a detailed analysis of the ESR spectra of (7,8)SA in terms of two components: one component arising from the labels surrounded exclusively by phospholipids, the other component arising from labels of reduced mobility perturbed by the vicinity of the proteins. These results support the persistence of lipidic domains in the endocytic vesicles despite the accumulation of receptors which follows their formation.  相似文献   

8.
R D Pates  D Marsh 《Biochemistry》1987,26(1):29-39
Lipid-protein interactions in bovine rod outer segment disk membranes have been studied by using a series of eight stearic acid spin-label probes which were labeled at different carbon atom positions in the chain. In randomly oriented membrane dispersions, the electron spin resonance (ESR) spectra of the C-8, C-9, C-10, C-11, C-12, C-13, and C-14 atom positional isomers all apparently consist of two components. One of the components corresponds closely to the spectra obtained from dispersions of the extracted membrane lipids, and the other, which is characterized by a considerably greater degree of motional restriction of the lipid chains, is induced by the presence of the protein. Digital subtraction has been used to separate the two components. The proportion of the motionally restricted lipid component is approximately constant, independent of the position of the spin-label group, and corresponds to 30-40% of the total spin-label spectral intensity. The hyperfine splitting of the outer maxima in the difference spectra of the motionally restricted component decreases, and concomitantly, the line widths increase with increasing temperature but change relatively little with increasing distance of the spin-label group from the polar head-group region. This indicates that the corresponding chain motions of the protein-interacting lipids lie in the slow-motion regime of spin-label ESR spectroscopy (tau R approximately 10(-8) S) and that the mobility of these lipids increases with increasing temperature but does not vary greatly along the length of the chain. The data from the hyperfine splittings also suggest the existence of a polarity gradient immediately adjacent to the protein surface, as observed in the fluid lipid regions of the membrane. The more fluid lipid component is only slightly perturbed relative to the lipids alone (for label positions 5-14, inclusive), indicating the presence of chain motions on the nanosecond time scale, and the spectra also reveal a similar polarity profile in both lipid and membrane environments. ESR spectra have also been obtained as a function of magnetic field orientation with oriented membrane samples. For the C-14 atom positional isomer, the motionally restricted component is observed to have a large hyperfine splitting, with the magnetic field oriented both parallel and perpendicular to the membrane normal. This indicates that the motionally restricted lipid chains have a broad distribution of orientations at this label position.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

9.
To investigate the physical mechanism by which melittin inhibits Ca-adenosine triphosphatase (ATPase) activity in sarcoplasmic reticulum (SR) membranes, we have used electron paramagnetic resonance spectroscopy to probe the effect of melittin on lipid-protein interactions in SR. Previous studies have shown that melittin substantially restricts the rotational mobility of the Ca-ATPase but only slightly decreases the average lipid hydrocarbon chain fluidity in SR. Therefore, in the present study, we ask whether melittin has a preferential effect on Ca-ATPase boundary lipids, i.e., the annular shell of motionally restricted lipid that surrounds the protein. Paramagnetic derivatives of stearic acid and phosphatidylcholine, spin-labeled at C-14, were incorporated into SR membranes. The electronic paramagnetic resonance spectra of these probes contained two components, corresponding to motionally restricted and motionally fluid lipids, that were analyzed by spectral subtraction. The addition of increasing amounts of melittin, to the level of 10 mol melittin/mol Ca-ATPase, progressively increased the fraction of restricted lipids and increased the hyperfine splitting of both components in the composite spectra, indicating that melittin decreases the hydrocarbon chain rotational mobility for both the fluid and restricted populations of lipids. No further effects were observed above a level of 10 mol melittin/mol Ca-ATPase. In the spectra from control and melittin-containing samples, the fraction of restricted lipids decreased significantly with increasing temperature. The effect of melittin was similar to that of decreased temperature, i.e., each spectrum obtained in the presence of melittin (10:1) was nearly identical to the spectrum obtained without melittin at a temperature approximately 5 degrees C lower. The results suggest that the principal effect of melittin on SR membranes is to induce protein aggregation and this in turn, augmented by direct binding of melittin to the lipid, is responsible for the observed decreases in lipid mobility. Protein aggregation is concluded to be the main cause of inactivation of the Ca-ATPase by melittin, with possible modulation also by the decrease in mobility of the boundary layer lipids.  相似文献   

10.
Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.  相似文献   

11.
The microwave saturation properties of various spin-labeled lipids in reconstituted complexes of the myelin proteolipid protein with dimyristoyl phosphatidylcholine have been studied both by conventional and saturation transfer electron spin resonance (ESR) spectroscopy. In the fluid phase, the conventional ESR spectra consist of a fluid and a motionally restricted (i.e., protein-associated) component, whose relative proportions can be determined by spectral subtractions and depend on the selectivity of the particular spin-labeled lipid for the protein. At 4 degrees C when the bulk lipid is in the gel phase, the integrated intensity of the saturation transfer ESR spectra displays a linear dependence on the fraction of motionally restricted lipid that is deduced from the conventional ESR spectra in the fluid phase, indicating the presence of distinct populations of free and protein-interacting lipid with no exchange between them on the saturation transfer ESR time scale in the gel phase. At 30 degrees C when the bulk lipid is in the fluid phase, the saturation transfer integral displays a nonlinear dependence on the fraction of motionally restricted lipid, consistent with exchange between the two lipid populations on the saturation transfer ESR time scale in the fluid phase. For lipid spin labels with different selectivities for the protein in complexes of fixed lipid/protein ratio, the data in the fluid phase are consistent with a constant (diffusion-controlled) on-rate for exchange at the lipid-protein interface. Values ranging between 1 and 9 x 10(6) s-1 are estimated for the intrinsic off-rates for exchange of spin-labeled stearic acid and phosphatidylcholine, respectively, at 30 degrees C. Conventional continuous wave saturation experiments lead to similar conclusions regarding the lipid exchange rates in the fluid and gel phases of the lipid/protein recombinants. The ESR saturation studies therefore demonstrate exchange on the time scale of the nitroxide spin-lattice relaxation at the lipid-protein interface of myelin proteolipid/dimyristoyl phosphatidylcholine complexes in the fluid phase but not in the gel phase.  相似文献   

12.
Gramicidin S was incorporated into dimyristoylphosphatidylcholine dispersions and the observed two-component EPR spectra of spin-labelled lipids at 30 °C were analysed by a two-stage algorithm, including spectral subtractions and two-site exchange simulations. A limited range of temperatures around 30 °C was found suitable for such measurements. It has been found that negatively charged labelled lipids display a selectivity towards the intramembranous part of the peptide. The relative association constants for spin-labelled stearic acid (14-SASL) and phosphatidylserine (14-PSSL) were K r = 2.08± 0.10 and 1.18±0.08, respectively, when compared with the zwitterionic phosphatidylcholine label (14-PCSL, K r≡ 1). The lateral diffusion of spin-labelled lipids in peptide-free regions causes exchange between those labels in the bulk fluid lipid phase and motionally restricted boundary labelled lipids at the apolar interface of gramicidin S. Owing to exchange, the spectral anisotropy of labelled lipids giving rise to the slow-motion spectral component was gradually decreased, and there was an augmentation of spectral intensity in between the motionally restricted (slow motion) and the fast tumbling (motionally averaged) labelled lipid components. Two-component exchange simulations allowed the determination of off-rates of labelled phospholipids, showing an inverse proportionality with lipid-protein selectivity. Spin-labelled procaine exhibited limited selectivity and fast exchange similar to the on-coming non-specifically associated lipids. Received: 25 May 1998 / Revised version: 14 September 1998 / Accepted: 5 November 1998  相似文献   

13.
The D-galactose-H(+) symport protein (GalP) of Escherichia coli is a homologue of the human glucose transport protein, GLUT1. After amplified expression of the GalP transporter in E. coli, lipid-protein interactions were studied in gradient-purified inner membranes by using spin-label electron paramagnetic resonance (EPR) spectroscopy. Phosphatidylethanolamine, -glycerol, -choline and -serine, in addition to phosphatidic and stearic acids, were spin-labelled at the 14 C-atom of the sn-2 chain. EPR spectra of these spin labels at probe amounts in GalP membranes consist of two components. One component corresponds to a lipid population whose motion is restricted by direct interaction with the transmembrane sections of the integral protein. The other component corresponds to a lipid population with greater chain mobility, and is similar to the single-component EPR spectrum of the spin-labelled lipids in membranes of E. coli lipid extract. Quantitation of the protein-interacting spin-label component allows determination of the stoichiometry and selectivity of lipid-protein interactions. On average, approximately 20 mol of lipid are motionally restricted per 52 kDa of protein in GalP membranes. At the pH of the transport assay, there is relatively little selectivity between the different phospholipids tested. Only stearic acid displays a stronger preferential interaction with this protein.  相似文献   

14.
M Esmann  K Hideg  D Marsh 《Biochemistry》1988,27(11):3913-3917
The interactions of a series of spin-labeled fatty acids, in which the nitroxide ring is incorporated in different ways as an integral part of the hydrocarbon chain, with the (Na+,K+)-ATPase in membranes from Squalus acanthias, have been studied by electron spin resonance spectroscopy. The fatty acids are 2,4-, 2,5-, and 3,2-substituents of 2,2,5,5-tetramethylpyrrolidine-N-oxyl and belong to the class of minimal perturbation nitroxide probes. For all five fatty acid labels, a motionally restricted lipid component was observed in the ESR spectra of (Na+,K+)-ATPase membranes, in addition to the fluid component, which was found in the spectra of the extracted membrane lipids. The pH dependence of the motionally restricted spin-label population indicated a sensitivity in the selectivity of the lipid-protein interaction to the protonation state of the fatty acid. These results agree with those found previously for the conventional oxazolidine (doxyl) fatty acid and phospholipid spin-label derivatives [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] and indicate that the motion of the lipid chains is significantly hindered by interaction with the protein, irrespective of the nature of the spin-label group.  相似文献   

15.
ESR spectra of spin probes were used to monitor lipid-protein interactions in native and cholesterol-enriched microsomal membranes. In both systems composite spectra were obtained, one characteristic of bulk bilayer organization and another due to a motionally restricted population, which was ascribed to lipids in a protein microenvironment. Computer spectral subtractions revealed that cholesterol modulates the order/mobility of both populations in opposite ways, i.e., while the lipid bilayer region gives rise to more anisotropic spectra upon cholesterol enrichment, the spectra of the motionally restricted population become indicative of increased mobility and/or decreased order. These events were evidenced by measurement of both effective order parameters and correlation times. The percentages of the motionally restricted component were invariant in native and cholesterol-enriched microsomes. Variable temperature studies also indicated a lack of variation of the percentages of both spectral components, suggesting that the motionally restricted one was not due to protein aggregation. The results correlate well with the effect of cholesterol enrichment on membrane-bound enzyme kinetics and on the behavior of fluorescent probes [Castuma & Brenner (1986) Biochemistry 25, 4733-4738]. Several hypothesis are put forward to explain the molecular mechanism of the cholesterol-induced spectral changes.  相似文献   

16.
B S Selinsky  P L Yeagle 《Biochemistry》1984,23(10):2281-2288
Phosphorus nuclear magnetic resonance spectra of sarcoplasmic reticulum membranes from rabbit muscle and of recombined membranes containing the calcium-dependent adenosinetriphosphatase (Ca-ATPase) of sarcoplasmic reticulum reveal two distinguishable, overlapping resonances. One resonance resembles a normal phospholipid bilayer resonance, and the other is much broader. The broader component is not seen in protein-free phospholipid vesicles. In recombined membranes of the Ca-ATPase, the intensity found in the broad component was proportional to the concentration of protein in the vesicles. The two-component spectra are interpreted to arise from at least two different domains of phospholipids, one of which is motionally restricted by the Ca-ATPase. Phospholipids exchange between these two domains at a rate less than 10(3) s-1. A model for protein-lipid interactions in membranes containing the Ca-ATPase is proposed in which some of the phospholipid head groups of the membrane interact directly with the protein.  相似文献   

17.
Lipid analysis and ESR studies were carried out on prostasomes isolated from human semen. Cholesterol plus phospholipids amounted to approximately 0.80 mumol per mg protein with a striking quantitative domination of cholesterol over the phospholipids, the molar ratios of cholesterol/sphingomyelin/glycerophospholipids being 4:1:1. Saturated and monounsaturated fatty acids were dominating both in the glycerophospholipids and in sphingomyelin. The order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes were very high, viz. 0.75 for 5-doxylstearic acid and 0.30 for 16-doxylstearic acid at 25 degrees C. Slightly lower values were obtained for the spin-labelled fatty acids when they were incorporated into dispersions of extracted prostasome lipids or into synthetic lipid mixtures of similar composition. The highly ordered lipids in the prostasome membrane thus seemed to be minimally perturbed by proteins in the membrane and ESR spectra showed no signs of immobilized lipids.  相似文献   

18.
Freely-diffusing phospholipid spin labels have been employed to study rhodopsin-lipid interactions in frog rod outer segment disc membranes. Examination of the ESR spectra leads us to the conclusion that there are two motionally distinguishable populations of lipid existing in frog rod outer segment membranes over a wide physiological temperature range. Each of the spin probes used shows a two-component electron spin resonance (ESR) spectrum, one component of which is motionally restricted on the ESR timescale, and represents between 33 and 40% of the total integrated spectral intensity. The second spectral component which accounts for the remainder of the spectral intensity possesses a lineshape characteristic of anisotropic motion in a lipid bilayer, very similar in shape to that observed from the same spin labels in dispersions of whole extracted frog rod outer segment lipid. The motionally restricted spectral component is attributed to those spin labels in contact with the surface of rhodospin, while the major component is believed to originate from spin labels in the fluid lipid bilayer region of the membranes. Calculations indicate that the motionally restricted lipid is sufficient to cover the protein surface. This population of lipids is shown here and elsewhere (Watts, A., Volotovski, I.D. and Marsh, D. (1979) Biochemistry 18, 5006-5013) to be by no means rigidly immobilized, having motion in the 20 ns time regime as opposed to motions in the one nanosecond time regime found in the fluid bilayer. Little selectivity for the motionally restricted population is observed between the different spin-labelled phospholipid classes nor with a spin-labelled fatty acid or sterol.  相似文献   

19.
Functional membranes containing purified Torpedo californica acetylcholine receptor and dioleoylphosphatidylcholine (DOPC) were prepared by a cholate dialysis procedure with lipid to protein ratios of 100-400 to 1 (mol/mol). Spin-labeled lipids were incorporated into the reconstituted membranes and into native membranes prepared from Torpedo electroplax, and electron paramagnetic resonance (EPR) spectra were recorded between 0 and 20 degrees C. The spin-labels included nitroxide derivatives of stearic acid (16-doxylstearic acid), androstane, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA). The phospholipid spin-labels had 16-doxylstearic acid in the sn-2 position. All the spectra showed two components corresponding to a relatively mobile bilayer component and a motionally restricted "protein-perturbed" component. The relative amounts of mobile and perturbed components were quantitated by spectral subtraction and integration techniques. The mobile/perturbed ratio was somewhat temperature dependent, and the results are discussed in terms of exchange between mobile and perturbed environments. Plots of the mobile/perturbed ratios vs. lipid/protein ratios at 1 degree C gave straight lines from which the relative binding affinity of each spin-label and the number of perturbed lipids per receptor protein could be calculated. All the spin-labels gave similar values for the number of perturbed lipids (40 +/- 7), a number close to the number of lipids that will fit around the intramembranous perimeter of the receptor. The affinities of the spin-labeled lipids for the receptor relative to DOPC were androstane (K = 4.3) congruent to 16-doxylstearic acid (4.1) greater than PA (2.7) greater than PE (1.1) approximately PC (1.0) approximately PS (0.7). The lipids having the highest affinity for the acetylcholine receptor were also those that have the largest effects on the ion flux functional properties of the receptor, and the results are discussed in terms of lipid effects on receptor function.  相似文献   

20.
The interaction of spin-labeled lipids with beta-barrel transmembrane proteins has been studied by the electron spin resonance (ESR) methods developed for alpha-helical integral proteins. The outer membrane protein OmpA and the ferrichrome-iron receptor FhuA from the outer membrane of Escherichia coli were reconstituted in bilayers of dimyristoylphosphatidylglycerol. The ESR spectra from phosphatidylglycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the beta-barrel, in addition to that from the fluid bilayer lipids. The stoichiometry of motionally restricted lipids, 11 and 32 lipids/monomer for OmpA and FhuA, respectively, is constant irrespective of the total lipid/protein ratio. It is proportional to the number of transmembrane beta-strands, eight for OmpA and 22 for FhuA, and correlates reasonably well with the intramembranous perimeter of the protein. Spin-labeled lipids with different polar headgroups display a differential selectivity of interaction with the two proteins. The more pronounced pattern of lipid selectivity for FhuA than for OmpA correlates with the preponderance of positively charged residues facing the lipids in the extensions of the beta-sheet and shorter interconnecting loops on the extracellular side of FhuA.  相似文献   

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