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1.
A new method for studying 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 of 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 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.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 445–448.Original Russian Text Copyright ¢ 2005 by Radchenko, Shuvaeva, Il’nitskaya, Tret’yakov, Lipkin. 相似文献
2.
Exchange rates and numbers of annular lipids for the calcium and magnesium ion dependent adenosinetriphosphatase 总被引:3,自引:0,他引:3
A spin-labeled phospholipid is used to study lipid-protein interactions in the (Ca2+,Mg2+)-ATPase of sarcoplasmic reticulum from muscle. A novel null method is used to decompose composite electron spin resonance spectra into two components, characteristic of immobilized and mobile environments. Calculations based on a random mixing model suggest that protein-protein interactions will be relatively rare in these systems and that the immobilized lipid does not represent lipid trapped between proteins but rather represents annular phospholipid at the lipid-protein interface of the adenosinetriphosphatase. The apparent decrease in the amount of immobilized lipid with increasing temperature is shown to be consistent with lipid exchange between bulk and annulus, characterized by an exchange time of 10(-7) s at 37 degrees C. A minimum number of annular phospholipid sites of 32 and 22 are calculated at 0 and 37 degrees C, respectively. 相似文献
3.
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
4.
5.
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
6.
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
7.
A model recently used to study lipid-protein interactions in one-component lipid bilayers (Sperotto and Mouritsen, 1991 a, b) has been extended in order to include two different lipid species characterized by different acyl-chain lengths. The model, which is a statistical mechanical lattice model, assumes that hydrophobic matching between lipid-bilayer hydrophobic thickness and hydrophobic length of the integral protein is an important aspect of the interactions. By means of Monte Carlo simulation techniques, the lateral distribution of the two lipid species near the hydrophobic protein-lipid interface in the fluid phase of the bilayer has been derived. The results indicate that there is a very structured and heterogeneous distribution of the two lipid species near the protein and that the protein-lipid interface is enriched in one of the lipid species. Out of equilibrium, the concentration profiles of the two lipid species away from the protein interface are found to develop a long-range oscillatory behavior. Such dynamic membrane heterogeneity may be of relevance for determining the physical factors involved in lipid specificity of protein function. 相似文献
8.
Tethered bilayer lipid membranes (tBLMs) are important tools for studying protein–lipid interactions. The widely used methodology for the preparation of these membranes is the fusion of phospholipid vesicles from an aqueous medium onto an anchored phospholipid layer. The preparation of phospholipid vesicles is a long and tedious procedure. There is another simple method, rapid solvent exchange, for preparing lipid membranes. However, there is a lack of information on the effects of the preparation method of tBLMs on their interactions with proteins. Therefore, we present in this paper a comparative study on the binding of lysozyme onto tBLMs prepared by the abovementioned methods. The prepared tBLMs have either zwitterionic or anionic characteristics. The results show that lysozyme binding onto the prepared tBLMs is unaffected by the preparation method of the tBLMs, suggesting that the tedious fusion method might be replaced by the simple rapid solvent exchange method without altering the level of protein–lipid interactions. 相似文献
9.
In aqueous solution bovine myelin basic protein exhibits no significant alpha-helical or beta-pleated sheet structure. However, in vivo this protein is associated largely with the myelin membrane: experiments have therefore been performed to determine the structure of the protein when bound to lipid bilayers. Circular dichroism spectra show that this protein undergoes a major conformational change on binding to lipid bilayer vesicles formed from diacylphosphatidylserine or diacylphosphatidic acid, and on binding to micelles of several detergents. Association with diacylphosphatidylcholine failed to induce a structural change: this observation is interpreted in terms of an earlier report that lysophosphatidylcholine does increase the alpha-helical content of basic protein. These circular dichroism measurements and studies of the binding to the bilayer-forming lipids appear to provide support for significant hydrophobic lipid-protein interactions. Similar studies using two peptides produced by cleavf basic protein indicate that a major structure-forming region in the middle of the protein has been disrupted by this scission. 相似文献
10.
《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
11.
12.
Gröger T Nathoo S Ku T Sikora C Turner RJ Prenner EJ 《Chemistry and physics of lipids》2012,165(2):216-224
A detailed understanding of biomembrane architecture is still a challenging task. Many in vitro studies have shown lipid domains but much less information is known about the lateral organization of membrane proteins because their hydrophobic nature limits the use of many experimental methods. We examined lipid domain formation in biomimetic Escherichia coli membranes composed of phosphatidylethanolamine and phosphatidylglycerol in the absence and presence of 1% and 5% (mol/mol) membrane multidrug resistance protein, EmrE. Monolayer isotherms demonstrated protein insertion into the lipid monolayer. Subsequently, Brewster angle microscopy was applied to image domains in lipid matrices and lipid-protein mixtures. The images showed a concentration dependent impact of the protein on lipid domain size and shape and more interestingly distinct coexisting protein clusters. Whereas lipid domains varied in size (14-47μm), protein clusters exhibited a narrow size distribution (2.6-4.8μm) suggesting a non-random process of cluster formation. A 3-D display clearly indicates that these proteins clusters protrude from the membrane plane. These data demonstrate distinct co-existing lipid domains and membrane protein clusters as the monofilm is being compressed and illustrate the significant mutual impact of lipid-protein interactions on lateral membrane architecture. 相似文献
13.
Interaction of soluble and membrane proteins with monolayers of glycosphingolipids. 总被引:2,自引:2,他引:0
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1. The interactions of four proteins (albumin, myelin basic protein, melittin and glycophorin) with eight neutral or acidic glycosphingolipids, including sulphatides and gangliosides, five zwitterionic or anionic phospholipids and some of their mixtures, were studied in lipid monolayers at the air/145 mM-NaCl interface. 2. In lipid-free interfaces, the surface pressure and surface potential reached by either soluble or integral membrane proteins did not reveal marked differences. 3. All the proteins studied showed interactions with each of the lipids but the maximal interactions were found for basic proteins with acidic glycosphingolipids. 4. Surface-potential measurements indicated that different dipolar organizations at the interface can be adopted by lipid-protein interactions showing the same value for surface free energy. 5. The individual surface properties of either the lipid of protein component are modified as a consequence of the lipid-protein interaction. 6. In mixed-lipid monolayers, the composition of the interface may affect the lipid-protein interactions in a non-proportional manner with respect to the relative amount of the individual lipid components. 相似文献
14.
Stella M. Lu 《Critical reviews in biochemistry and molecular biology》2018,53(2):192-207
The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid–protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the “lipid raft” concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms “membrane raft” or “membrane nanodomain” are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae. 相似文献
15.
Fluorescence correlation spectroscopy (FCS) was applied to examine the interactions between a protein and a membrane lipid. The protein 4.1-phosphatidyl serine (PS) interactions served as the model system to demonstrate the membrane lipid-protein interactions. This protein was labeled with rhodamine and its interactions with PS-liposomes were measured by FCS. The present results clearly demonstrated that a small protein molecule, protein 4.1, interacts specifically with a large particle, a PS-liposome. This interaction appears to be hydrophobic and not electrostatic, since the bound protein 4.1 did not dissociate in solution and was specifically released from PS-liposomes by treatment with phospholipase A(2) (PLA(2)). In the present study, using FCS we could demonstrate that the serine residue of PS is required for protein 4.1 to bind to PS-liposomes and that the bound protein 4.1 is closely associated with the fatty acid of the PS molecule in the liposomes. 相似文献
16.
In this article, we examine the mechanical role of the lipid bilayer in ion channel conformation and function with specific reference to the case of the mechanosensitive channel of large conductance (MscL). In a recent article we argued that mechanotransduction very naturally arises from lipid-protein interactions by invoking a simple analytic model of the MscL channel and the surrounding lipid bilayer. In this article, we focus on improving and expanding this analytic framework for studying lipid-protein interactions with special attention to MscL. Our goal is to generate simple scaling relations which can be used to provide qualitative understanding of the role of membrane mechanics in protein function and to quantitatively interpret experimental results. For the MscL channel, we find that the free energies induced by lipid-protein interaction are of the same order as the measured free energy differences between conductance states. We therefore conclude that the mechanics of the bilayer plays an essential role in determining the conformation and function of the channel. Finally, we compare the predictions of our model to experimental results from the recent investigations of the MscL channel by a variety of investigators and suggest a suite of new experiments. 相似文献
17.
Lipophilin, a hydrophobic protein purified from the proteolipid of normal human brain myelin, was recombined with phosphatidylcholine by solubilization of the lipid and protein in 2-chloro-ethanol followed by dialysis against buffer. This method resulted in homogeneous incorporation of the protein into lipid vesicles as judged by sedimentation on a sucrose gradient and freeze fracture electron microscopy. The lipid-protein vesicles were single layered, 1000–2000 Å in diameter and the freeze fracture faces contained intramembrane particles. The effect of lipophilin on the organization of the lipid was studied by use of spin label probes. Two distinct components were present in the spectrum of fatty acid spin labels in the lipid-protein vesicles. One was immobilized presumably due to the presence of boundary lipid around the protein and the second component was indicative of aniostropic motion similar to the spectrum in phosphatidylcholine vesicles and probably due to a lamellar phase but with a slightly greater order parameter. Lipophilin was found to increase the order parameter linearly with increasing concentration of protein incorporated into the vesicles. However, the phase transition temperature as measured from the 2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPO) solubility parameter was unchanged. 相似文献
18.
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
19.
R Brasseur 《The Journal of biological chemistry》1991,266(24):16120-16127
Several types of lipid-associating helices exist: transmembrane helices such as in receptor proteins, pore-forming helices in ion channel proteins, fusion-inducing peptides in viral proteins, and amphipathic helices such as in plasma apolipoproteins. In order to propose a classification of these helices according to their molecular properties, we introduce the concept of molecular hydrophobicity potential for such helical segments. The calculation of this parameter for alpha-helices enables the visualization of the hydrophobic and hydrophilic envelopes around the peptide and their three-dimensional representation by molecular graphics. We have used this parameter to differentiate between pore-forming helices with a hydrophobic envelope larger than the hydrophilic component, membrane-spanning helices surrounded almost entirely by an hydrophobic envelope, fusiogenic peptides with an hydrophobicity gradient both around the helix and along the axis, and finally, amphipathic helices with a predominantly hydrophilic envelope. The structure of the lipid-protein complexes is determined by a number of different interactions: the hydrophobic interaction of the apolar faces of the helices with lipids, the polar interaction of the hydrophilic sides of different helices with each other, and the interaction of hydrophilic residues with the aqueous solvent. The relative magnitude of the hydrophobic and hydrophilic envelopes accounts for the differences in the structure of the lipid-protein complexes. Purely hydrophobic interactions stabilize transmembrane helical segments, while hydrophobic interactions with the lipid phase and with each other are involved in the stabilization of the pore-forming helices. In contrast, both hydrophobic interactions with the lipids and hydrophilic interactions with the aqueous phase contribute to the arrangement of amphipathic helices around the edges of the discoidal lipid-apoprotein complexes. 相似文献
20.
The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second-order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision-induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions. 相似文献