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1.
Riadchenko VV Shuvaeva TM Il'nitskaia EV Tret'iakov VE Lipkin VM 《Bioorganicheskaia khimiia》2005,31(4):445-448
A new method for studying the lipid-protein interactions in vitro is developed. It enables the study of the transporting activity of a protein toward a lipid ligand, including the case with an unknown lipid type. The method can be considered as a variant of partition three-phase chromatography with two stationary (donor and acceptor) phases and one mobile phase. The protein under study is dissolved in an aqueous mobile phase and induces a specific delivery of a lipid to the acceptor lipid layer. The transported lipid is identified in the Folch lipid extracts from the acceptor layer and aqueous phase. The secretory protein with M 45 kDa from the rat olfactory epithelium is shown to be a carrier of phosphatidylinositol 3,4,5-triphosphate. Our approach opens up new possibilities in the study of lipid-protein interactions in vitro and has a number of advantages over the methods now used for these purposes. 相似文献
2.
The effect of temperature on native microsomal membrane vesicles isolated from Tetrahymena is investigated by wide angle X-ray diffraction. A 4.2 Å reflection, typical for lipids in the crystalline state, can be recorded in the temperature range between 0°C and 35°C. Quantitative evaluation of this reflection reveals a broad thermotropic ‘two-stage’ liquid crystallinecrystalline lipid phase separation with a ‘breakpoint’ at approx. 18°C. This ‘breakpoint’ coincides with the emergence of lipid-protein segregations in endomembranes of intact Tetrahymena cells as previously visualized by freeze-etch electron microscopy. 相似文献
3.
Lipid A was obtained in a high yield (27%) by the hydrolysis of lipopolysaccharide from the marine gamma proteobacterium Marinomonas communis ATCC 27118T with 1% AcOH. Using chemical analysis and 1D and 2D NMR spectroscopic and fast atom bombardment mass spectrometric methods, it was shown to be β-1′,6-linked D-glucosaminobiose 1-phosphate acylated with (R)-3-dodecanoyl- or (R)-3-decanoyloxydecanoic acid, (R)-3-{(R)-3-hydroxydecanoyloxy)]decanoic acid and (R)-3-hydroxydecanoic acid at the C2, C2′ and C3 positions, respectively. Uncommon structural peculiarities (a low acylation and phosphorylation degree) of the M .communis lipid A in comparison with those of terrestrial bacteria may be of pharmacological interest. The potential physiological meaning of this lipid A and compounds of similar structure are discussed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 404–413.Original Russian Text Copyright © 2005 by Vorob’eva, A. Dmitrenok, P. Dmitrenok, Isakov, Krasikova, Solov’eva.The article was translated by the authors. 相似文献
4.
A comparative spin-label study of isolated plasma membranes and plasma membranes of whole cells and protoplasts from cold-hardened and nonhardened winter rye 总被引:1,自引:0,他引:1
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Windle JJ 《Plant physiology》1988,88(4):1388-1396
Lipid-lipid and lipid-protein interactions in the plasma membranes of whole cells and protoplasts and an isolated plasma membrane fraction from winter rye (Secale cereale L. cv Puma) have been studied by spin labeling. Spectra were recorded between −40°C and 40°C using the freely diffusing spin-label, 16-doxyl stearic acid, as a midbilayer membrane probe. The probe was reduced by the whole cells and protoplasts and reoxidized by external potassium ferricyanide. The reoxidized probe was assumed to be localized in the plasma membrane. The spectra consisted of the superposition of a narrow and a broad component indicating that both fluid and immobilized lipids were present in the plasma membrane. The two components were separated by digital subtraction of the immobilized component. Temperature profiles of the membranes were developed using the percentage of immobilized lipid present at each temperature and the separation between the outermost hyperfine lines for the fluid lipid component. Lipid immobilization was attributed to lipid-protein interactions, lipid-cell wall interactions, and temperature-induced lipid phase transitions to the gel-state. Temperature profiles were compared for both cold-hardened and nonhardened protoplasts, plasma membranes, and plasma membrane lipids, respectively. Although cold-hardening extended the range of lipid fluidity by 5°C, it had no effect on lipid-protein interactions or activation energies of lipid mobility. Differences were found, however, between the temperature profiles for the different samples, suggesting that alterations in the plasma membrane occurred as a consequence of the isolation methods used. 相似文献
5.
Aromatic amino acids of membrane proteins are enriched at the lipid-water interface. The role of tryptophan on the folding and stability of an integral membrane protein is investigated with ultraviolet resonance Raman and fluorescence spectroscopy. We investigate a model system, the β-barrel outer membrane protein A (OmpA), and focus on interfacial tryptophan residues oriented toward the lipid bilayer (trp-7, trp-170, or trp-15) or the interior of the β-barrel pore (trp-102). OmpA mutants with a single tryptophan residue at a nonnative position 170 (Trp-170) or a native position 7 (Trp-7) exhibit the greatest stability, with Gibbs free energies of unfolding in the absence of denaturant of 9.4 and 6.7 kcal/mol, respectively. These mutants are more stable than the tryptophan-free OmpA mutant, which exhibits a free energy of unfolding of 2.6 kcal/mol. Ultraviolet resonance Raman spectra of Trp-170 and Trp-7 reveal evolution of a hydrogen bond in a nonpolar environment during the folding reaction, evidenced by systematic shifts in hydrophobicity and hydrogen bond markers. These observations suggest that the hydrogen bond acceptor is the lipid acyl carbonyl group, and this interaction contributes significantly to membrane protein stabilization. Other spectral changes are observed for a tryptophan residue at position 15, and these modifications are attributed to development of a tryptophan-lipid cation-π interaction that is more stabilizing than an intraprotein hydrogen bond by ∼2 kcal/mol. As expected, there is no evidence for lipid-protein interactions for the tryptophan residue oriented toward the interior of the β-barrel pore. These results highlight the significance of lipid-protein interactions, and indicate that the bilayer provides more than a hydrophobic environment for membrane protein folding. Instead, a paradigm of lipid-assisted membrane protein folding and stabilization must be adopted. 相似文献
6.
Reconstituted membrane systems of synthetic phosphatidylcholines and the integral membrane enzyme cytochrome c oxidase were prepared in order to conduct nuclear magnetic resonance studies of lipid-protein interactions. These lipids, labeled with a geminate difluoro group on the 1-position hydrocarbon chain, were combined with the enzyme to give active lipid-protein particles with a well-defined ratio of lipid to protein. The fluorine magnetic resonance spectra of a series of preparations with different lipid/protein ratios suggest that the hydrocarbon chain mobility of the lipid is substantially reduced with increasing amounts of protein. The fluorine spectra of a single lipid-protein preparation show a dramatic increase in the number of the more mobile lipid chains with increasing temperature. The results suggest that the enzyme orders the lipid bilayer well beyond those lipids in direct contact with the protein surface, and that the amount of the lipid restricted by the enzyme is dependent upon temperature. The exchange of lipid between the restricted and the more mobile lipid environments most probably does not occur over the time scale measurable by the magnetic resonance techniques, about 10(-3) s. 相似文献
7.
8.
David R. Garmer 《Journal of biomolecular structure & dynamics》2013,31(5):525-546
Abstract This work presents MD calculations on a model 5-HT2A G protein-coupled receptor embedded in DOPC membrane bilayers at different lipid:protein ratios. The primary purpose is to evaluate physical properties of the system to probe membrane dynamics and the solvation interactions. This showed several kinds of apparent cooperativity phenomena in distributions, lipid dynamics, and hydrogen bond interactions. The integral protein appears to cause a disordered condensation of the local lipid structure which does not extend far beyond the surface layer of lipids. This, and changes in the lipid-protein interaction profiles give a start toward understanding membrane-protein selectivity. The presence of a partially immobilized surface layer of lipids may generally slow down kinetics of solute-solute interactions in similar liquid-crystal membranes. A hydration algorithm was also applied to the GPCR model producing a detailed water structure in some of the internal cavities, including in the areas proposed for ligand binding. Other properties examined included the distributions of lipid groups, the membrane electrostatic potential, and some of the short-time protein dynamics. 相似文献
9.
Piccotti L Buratta M Giannini S Gresele P Roberti R Corazzi L 《The Journal of membrane biology》2004,198(1):43-53
Factors influencing the release and anchorage of cytochrome c to the inner membrane of brain mitochondria have been investigated. Metabolic activity of mitochondria caused a decrease in the membrane potential Δψm, accompanied by detachment of the protein from the inner membrane. In a model system of cytochrome c reconstituted in cardiolipin (CL) liposomes, phosphate was used to breach the hydrophilic lipid-protein interactions. About 44% cytochrome c was removable when heart CL (80% 18:2n-6) was employed, whereas the remaining protein accounted for the tightly bound conformation characterized by hydrophobic lipid-protein interactions. Cytochrome c release from brain CL liposomes was higher compared to heart CL, consistent with lower polyunsaturated fatty acid content. The release was even higher with CL extracted from metabolically stressed mitochondria, exhibiting more saturated fatty acid profile compared to control (30% vs.17%). Therefore, weakening of the hydrophobic interactions due to saturation of CL may account for the observed cytochrome c release from mitochondria following metabolic stress. Moreover, mitochondria enriched with polyunsaturated CL exhibited higher Δψm, compared to less unsaturated species, suggesting that CL fatty acid composition influences Δψm. Mitochondria incorporated exogenous cytochrome c without protease-sensitive factors or Δψm. The internalized protein anchored to the inner membrane without producing swelling, as monitored by forward and side light scattering, but produced Δψm consumption, suggesting recovery of respiratory activity. The Δψm decrease is ascribed to a selected mitochondrial population containing the incorporated cytochrome c.This revised version was published online in August 2005 with a corrected cover date. 相似文献
10.
Dov Lichtenberg 《生物化学与生物物理学报:生物膜》1985,821(3)
This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, Re. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the ‘equilibrium partition model’, the dependence of the ‘solubilizing detergent concentration’ on the lipid concentration intersects with the lipid axis at −1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical (‘solubilizing’) effective ratio depends upon the nature of detergent less than any of these two factors. 相似文献
11.
《生物化学与生物物理学报:生物膜》1986,862(1):49-56
The influence of the liquid-expanded or liquid-condensed state of the lipid interface induced by changes of temperature on the lipid-protein interactions and their two-dimensional miscibility was studied for mixtures of melittin with different phospholipids (DPPC, DMPC, DOPC egg PC) and gangliosides (GM1, GD1a) in mixed monolayers at the air/145 mM NaCl interface. The critical amount of melittin at which a phase separation takes place in the mixed film increases as the glycosphingolipid or phospholipid is more liquid-expanded. The lipid-protein interaction increases the stability of both melittin and the lipid. The interaction of melittin with gangliosides is thermodynamically more favorable as these are more liquid-expanded. The interaction of melittin with phospholipids, on the other hand, is more favorable when the lipids are in the liquid-condensed state even if these films show lateral immiscibility at a lower proportion of protein compared to lipids in the liquid-expanded state. Hydration-dehydration effects in the polar head group region are likely to participate in these lipid-protein interactions. 相似文献
12.
Lipid phase transitions in Escherichia coli membranes and in dispersions of the extracted lipids were studied using the negatively charged fluorescence probe 1-anilinonaphthalene-8-sulfonate (ANS−) and the hydrophobic fluorescence probe N-phenyl-1-naphthylamine (NPN). The fluorescence change, ΔI, at the phase transition approaches a limiting value (ΔI)lim with increasing dye concentration. A comparison of the limiting values (Δ)limNPN obtained for membranes and the lipid standard allows us to estimate the lipid fraction, ρ, in the membrane that takes part in the phase transition (ρ = 80%). The same procedure carried out with ANS− yields a value of 42.5% for the lipid fraction that is accessible from the aqueous phase. These values, combined with published freeze-etching data for the particle density within the fracture plane of membranes are used to quantify the Davson-Danielli-Robertson-Benson-Singer membrane model which assumes a fluid lipid bilayer with “integral” proteins embedded in the lipid matrix and surface proteins attached to the lipid head groups. It appears that on the average one “integral” membrane protein is surrounded by about 600 lipid molecules and that about 130 of these molecules are closely coupled to the protein molecule, forming an halo in which the chain-chain interaction between the lipids is disturbed. About half of the bilayer surface is covered with proteins; part of these seem to be stacked. 相似文献
13.
(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l−1 (PDC activity 2.5 U ml−1), PAC levels of 103 g l−1 in the octanol phase and 12.8 g l−1 in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U−1 h−1) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l−1 h−1) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g−1).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”. 相似文献
14.
We present a theoretical study of the effect of different types of lipid-protein interactions on the thermodynamic properties of protein-containing lipid bilayers. The basis of this work is a theoretical model for pure lipid bilayer phase transitions developed earlier by Scott. Simple assumptions on the nature of the lipid conformations near a protein strongly affect the predicted properties of the model. Here we consider (a) random protein-lipid contacts, (b) enhanced contact between protein and lipid with a number of gauche bonds, and (c) enhanced contact between protein and all-trans but tilted lipid chains. Comparison of predicted results with experimental data seems to favor point c above but, by itself point c does not work well at larger protein concentrations. The results are discussed in the light of spectroscopic data, lipid-protein (plus annular lipid) miscibility, and interprotein forces. 相似文献
15.
M. Luisa Molina A. Marcela Giudici José A. Poveda Gregorio Fernández-Ballester Estefanía Montoya M. Lourdes Renart Asia M. Fernández José A. Encinar Gloria Riquelme Andrés Morales José M. González-Ros 《The Journal of biological chemistry》2015,290(42):25745-25755
There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp67–Glu71–Asp80 inactivation triad within the channel structure and its bearing on the selectivity filter. 相似文献
16.
To obtain insight into the potential role of the cytoskeleton on lipid mixing behavior in plasma membranes, the current study explores the influence of physisorbed actin filaments (F-actin) on lipid–lipid phase separations in planar model membrane systems containing raft-mimicking lipid mixtures of well-defined compositions using a complementary experimental approach of epifluorescence microscopy, fluorescence anisotropy, wide-field single molecule fluorescence microscopy, and interfacial rheometry. In particular, we have explored the impact of F-actin on cholesterol (CHOL)–phospholipid interactions, which are considered important for the formation of CHOL-enriched lipid raft domains. By using epifluorescence microscopy, we show that physisorbed filamentous actin (F-actin) alters the domain size of lipid–lipid phase separations in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol (CHOL). In contrast, no actin-induced modification in lipid–lipid phase separations is observed in the absence of POPS or when POPS is replaced by another anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Wide-field single molecule fluorescence microscopy on binary lipid mixtures indicate that PS and PG lipids show similar electrostatic interactions with physisorbed actin filaments. Complementary fluorescence anisotropy experiments on binary PS lipid-containing lipid mixtures are provided to illustrate the actin-induced segregation of anionic lipids. The similarity of electrostatic interactions between actin and both anionic lipids suggests that the observed differences in actin-mediated perturbations of lipid phase separations are caused by distinct PS lipid–CHOL versus PG lipid–CHOL interactions. We hypothesize that the actin cytoskeleton and some peripheral membrane proteins may alter lipid–lipid phase separations in plasma membranes in a similar way by interacting with PS lipids. 相似文献
17.
Wetting and capillary condensation as means of protein organization in membranes. 总被引:1,自引:0,他引:1
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Wetting and capillary condensation are thermodynamic phenomena in which the special affinity of interfaces to a thermodynamic phase, relative to the stable bulk phase, leads to the stabilization of a wetting phase at the interfaces. Wetting and capillary condensation are here proposed as mechanisms that in membranes may serve to induce special lipid phases in between integral membrane proteins leading to long-range lipid-mediated joining forces acting between the proteins and hence providing a means of protein organization. The consequences of wetting in terms of protein aggregation and protein clustering are derived both within a simple phenomenological theory as well as within a concrete calculation on a microscopic model of lipid-protein interactions that accounts for the lipid bilayer phase equilibria and direct lipid-protein interactions governed by hydrophobic matching between the lipid bilayer hydrophobic thickness and the length of the hydrophobic membrane domain. The theoretical results are expected to be relevant for optimizing the experimental conditions required for forming protein aggregates and regular protein arrays in membranes. 相似文献
18.
The bone marrow myelopeptide MP-2 (Leu-Val-Val-Tyr-Pro-Trp), exhibiting antitumor activity, and its retro-analogue (Trp-Pro-Tyr-Val-Val-Leu) were synthesized, and their properties were studied. The in vitro and in vivo activities of retro-MP-2 were comparable with those of MP-2. Both peptides equally restored the functional activity of T-lymphocytes inhibited by toxins released by HL-60 cells and inhibited by 70–82% the growth of various types of transplantable solid tumors: Ca-755 adenocarcinoma of the mammary gland, Lewis adenocarcinoma of the lung, and S180 sarcoma. The positions and intensities of the Cotton effects in CD spectra of the MP-2 peptide and its retro-analogue in various solvents are almost indistinguishable. The positions of extrema and integral intensities of the amide I and amide A bands in IR spectra of both peptides were practically identical.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 239–244.Original Russian Text Copyright © 2005 by Fonina, Ovchinnikov, Gur’yanov, Sychev, Belevskaya, Treshchalina. 相似文献
19.
David Sklan William S. Blaner Noriaki Adachi John Edgar Smith DeWitt S. Goodman 《Archives of biochemistry and biophysics》1982,214(1):35-44
A fluorescent, high-molecular-weight, lipid-protein aggregate was partially isolated from the cytosol fraction of rat liver by gel filtration on columns of Sepharose 4B or 6B. This aggregate was composed of approximately equal parts of protein and of lipid (mainly triglycerides), and was found to contain approximately 19% of the total liver vitamin A (predominantly as retinyl esters). Most of the liver cellular retinol-binding protein (CRBP) was found associated with the fluorescent, lipid-protein aggregate, along with much of the retinyl palmitate hydrolase activity present in the liver cytosol. The lipid-protein aggregate, and its several vitamin A-related components, all displayed an apparent hydrated density between 1.052 and 1.090 in the ultracentrifuge. CRBP in association with the lipid-protein aggregate was not immunoreactive in the CRBP radioimmunoassay. CRBP was, however, released from this aggregate and rendered immunoreactive by addition of detergents (e.g., Triton X-100). Three other lipid hydrolytic activities were also found in association with the lipid-protein aggregate, namely, triolein, cholesteryl oleate, and dipalmitoyl phosphatidylcholine hydrolase activities. These several hydrolytic activities were all found to be stimulated optimally by the addition of either sodium cholate or bovine serum albumin. With the information available, it is not clear whether this lipid-protein aggregate is formed in vitro, during liver homogenization, or whether it represents a specific lipoprotein with a significant functional role that exists in vivo in the liver cell. 相似文献
20.
The membrane lipid environment and lipid signaling pathways are potentially involved in the modulation of the activity of the cardiac Na+-Ca2+ exchanger (NCX). In the present study biophysical mechanisms of interactions of amphiphiles with the NCX and the functional consequences were examined. For this purpose, intracellular Ca2+ concentration jumps were generated by laser-flash photolysis of caged Ca2+ in guinea-pig ventricular myocytes and Na+-Ca2+ exchange currents (INa/Ca) were recorded in the whole-cell configuration of the patch-clamp technique. The inhibitory effect of amphiphiles increased with the length of the aliphatic chain between C7 and C10 and was more potent with cationic or anionic head groups than with uncharged head groups. Long-chain cationic amines (C12) exhibited a cut-off in their efficacy in INa/Ca inhibition. Analysis of the time-course, comparison with the Ni2+-induced INa/Ca block and confocal laser scanning microscopy experiments with fluorescent lipid analogs (C6- and C12-NBD-labeled analogs) suggested that amphiphiles need to be incorporated into the membrane. Furthermore, NCX block appears to require transbilayer movement of the amphiphile to the inner leaflet (“flip”). We conclude that both, hydrophobic and electrostatic interactions between the lipids and the NCX may be important factors for the modulation by lipids and could be relevant in cardiac diseases where the lipid metabolism is altered.This revised version was published online in August 2005 with a corrected cover date. 相似文献