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1.
Effect of acceptor membrane phosphatidylcholine on the catalytic activity of bovine liver phosphatidylcholine transfer protein 总被引:1,自引:0,他引:1
Protein-mediated transfer of phosphatidylcholine (PC) by bovine liver phosphatidylcholine transfer protein (PC-TP) was examined using a vesicle-vesicle assay system. Donor and acceptor membranes were prepared from Escherichia coli phospholipids and limiting amounts of egg yolk PC. PC transfer between vesicles of E. coli lipid/egg PC was markedly higher than transfer of PC from vesicles of E. coli lipid/egg PC to vesicles of E. coli lipid. Kinetic parameters of the interaction between PC-TP and E. coli lipid vesicles with or without PC was investigated. The apparent dissociation constants of the complex formed between PC-TP and these vesicles were determined kinetically and from double-reciprocal plots of intrinsic PC-TP fluorescence intensity increase versus vesicle concentration. The magnitude of the dissociation constant decreased as the PC content of the vesicles increased from 0 to 5 mol%. In addition, kinetic analysis revealed that the presence of PC in acceptor vesicles increased both the association and dissociation of PC-TP from vesicles. The effect of membrane PC molecules on transfer rates was examined using bis-phosphatidylcholine, a dimeric PC molecule which is not transferred by PC-TP. Rates of PC transfer to acceptor vesicles comprised of E. coli lipid/bis-PC were virtually identical to rates observed with acceptors vesicles prepared from E. coli lipid. The results suggest that transfer of PC by PC-TP is enhanced only when insertion of protein-bound PC occurs concurrently with the extraction of a molecule of membrane PC, i.e., a concerted, one-step catalytic mechanism for phospholipid exchange. 相似文献
2.
Alena Khmelinskaia Jonas Mücksch Franco Conci Grzegorz Chwastek Petra Schwille 《Biophysical journal》2018,114(10):2444-2454
In vitro membrane model systems are used to dissect complex biological phenomena under controlled unadulterated conditions. In this context, lipid monolayers are a powerful tool to particularly study the influence of lipid packing on the behavior of membrane proteins. Here, monolayers deposited in miniaturized fixed area-chambers, which require only minute amounts of protein, were used and shown to faithfully reproduce the characteristics of Langmuir monolayers. This assay is ideally suited to be combined with single-molecule sensitive fluorescence correlation spectroscopy (FCS) to characterize diffusion dynamics. Our results confirm the influence of lipid packing on lipid mobility and validate the use of FCS as an alternative to conventional surface pressure measurements for characterizing the monolayer. Furthermore, we demonstrate the effect of lipid density on the diffusional behavior of membrane-bound components. We exploit the sensitivity of FCS to characterize protein interactions with the lipid monolayer in a regime in which the monolayer physical properties are not altered. To demonstrate the potential of our approach, we analyzed the diffusion behavior of objects of different nature, ranging from a small peptide to a large DNA-based nanostructure. Moreover, in this work we quantify the surface viscosity of lipid monolayers. We present a detailed strategy for the conduction of point FCS experiments on lipid monolayers, which is the first step toward extensive studies of protein-monolayer interactions. 相似文献
3.
Toshihiko Sugiki Hideo Takahashi Michiko Nagasu Ichio Shimada 《Analytical biochemistry》2010,399(2):162-167
Intracellular lipid translocation is mediated by lipid transfer proteins and their functional impairments cause severe disorder in lipid metabolism. However, molecular mechanisms of protein-mediated lipid transfer remain unclear since conventional assay methods could not observe elementary processes in the lipid transfer reaction, such as lipid bilayer binding and lipid uptake. In this study, we found that ceramide extraction mediated by a ceramide trafficking protein (CERT) could be detected as decreasing the response of surface plasmon resonance (SPR). Based on this finding, we developed a novel real-time assay method that enables quantitative evaluation of the ceramide extraction activity of CERT, using the SPR technique. Performing this SPR-based assay using ceramide-embedded and ceramide-free lipid bilayers as ligands allows for the exclusive investigation of ceramide uptake processes, differentiating them from other CERT-membrane binding events. Furthermore, mutagenesis experiments of CERT using this SPR-based assay clearly elucidated whether an amino acid residue plays a role in the ceramide uptake process or the lipid bilayer binding process. This SPR-based assay method can separately evaluate the lipid extraction activity and lipid bilayer binding activity of the lipid transfer proteins, and provide more detailed information about lipid transfer phenomena. 相似文献
4.
Smith AJ Sanders MA Thompson BR Londos C Kraemer FB Bernlohr DA 《The Journal of biological chemistry》2004,279(50):52399-52405
Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 microm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet. 相似文献
5.
Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex 总被引:18,自引:0,他引:18
J R Wetterau K A Combs S N Spinner B J Joiner 《The Journal of biological chemistry》1990,265(17):9800-9807
A bovine liver protein which catalyzes the transfer of triglyceride between membranes has previously been isolated from the lumen of the microsomal fraction. When further purified about 100-fold, two polypeptides of molecular mass 58,000 and 88,000 were identified (Wetterau, J. R., and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38, 205-222). We demonstrate here that the two polypeptides (referred to as 58-kDa and 88-kDa, respectively) are associated in a protein-protein complex, and that the triglyceride transfer activity is associated with this complex. Antibodies specific for either polypeptide immunoprecipitated both the 58-kDa and 88-kDa polypeptides as well as the lipid transfer activity. The 58-kDa subunit of the microsomal transfer protein complex was identified as protein disulfide-isomerase (PDI) (EC 5.3.4.1) by 1) a comparison of the amino-terminal sequence of PDI and the 58-kDa subunit of the transfer protein, 2) a comparison of the reverse phase high performance liquid chromatography peptide maps of CNBr digests of PDI and the lipid transfer protein, 3) immunoprecipitation competition experiments in which PDI was found to compete with the lipid transfer protein for immunoprecipitation by the anti-58-kDa polyclonal antibodies, 4) immunological cross-reactivity of the microsomal triglyceride transfer protein complex with polyclonal antibodies raised against PDI, and 5) the appearance of protein disulfide isomerase activity following the dissociation of purified microsomal transfer protein complex with guanidine HCl. In conclusion, the microsomal triglyceride transfer protein has a multi-subunit structure which is unique compared to other intracellular lipid transfer proteins which have been described to be single polypeptides. The unexpected finding that PDI is a component of the microsomal triglyceride transfer protein complex suggests a new previously undescribed role for protein disulfide isomerase. 相似文献
6.
Morley S Cecchini M Zhang W Virgulti A Noy N Atkinson J Manor D 《The Journal of biological chemistry》2008,283(26):17797-17804
alpha-Tocopherol is a member of the vitamin E family that functions as the principal fat-soluble antioxidant in vertebrates. Body-wide distribution of tocopherol is regulated by the hepatic alpha-tocopherol transfer protein (alphaTTP), which stimulates secretion of the vitamin from hepatocytes to circulating lipoproteins. This biological activity of alphaTTP is thought to stem from its ability to facilitate the transfer of vitamin E between membranes, but the mechanism by which the protein exerts this activity remains poorly understood. Using a fluorescence energy transfer methodology, we found that the rate of tocopherol transfer from lipid vesicles to alphaTTP increases with increasing alphaTTP concentration. This concentration dependence indicates that ligand transfer by alphaTTP involves direct protein-membrane interaction. In support of this notion, equilibrium analyses employing filtration, dual polarization interferometry, and tryptophan fluorescence demonstrated the presence of a stable alphaTTP-bilayer complex. The physical association of alphaTTP with membranes is markedly sensitive to the presence of vitamin E in the bilayer. Some naturally occurring mutations in alphaTTP that cause the hereditary disorder ataxia with vitamin E deficiency diminish the effect of tocopherol on the protein-membrane association, suggesting a possible mechanism for the accompanying pathology. 相似文献
7.
The structure of "defective in induced resistance" protein of Arabidopsis thaliana, DIR1, reveals a new type of lipid transfer protein 总被引:1,自引:0,他引:1
Lascombe MB Bakan B Buhot N Marion D Blein JP Larue V Lamb C Prangé T 《Protein science : a publication of the Protein Society》2008,17(9):1522-1530
Screening of transfer DNA (tDNA) tagged lines of Arabidopsis thaliana for mutants defective in systemic acquired resistance led to the characterization of dir1-1 (defective in induced resistance [systemic acquired resistance, SAR]) mutant. It has been suggested that the protein encoded by the dir1 gene, i.e., DIR1, is involved in the long distance signaling associated with SAR. DIR1 displays the cysteine signature of lipid transfer proteins, suggesting that the systemic signal could be lipid molecules. However, previous studies have shown that this signature is not sufficient to define a lipid transfer protein, i.e., a protein capable of binding lipids. In this context, the lipid binding properties and the structure of a DIR1-lipid complex were both determined by fluorescence and X-ray diffraction. DIR1 is able to bind with high affinity two monoacylated phospholipids (dissociation constant in the nanomolar range), mainly lysophosphatidyl cholines, side-by-side in a large internal tunnel. Although DIR1 shares some structural and lipid binding properties with plant LTP2, it displays some specific features that define DIR1 as a new type of plant lipid transfer protein. The signaling function associated with DIR1 may be related to a specific lipid transport that needs to be characterized and to an additional mechanism of recognition by a putative receptor, as the structure displays on the surface the characteristic PxxP structural motif reminiscent of SH3 domain signaling pathways. 相似文献
8.
A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes 总被引:3,自引:0,他引:3
A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function. 相似文献
9.
J L Ellsworth M L Kashyap R L Jackson J A Harmony 《Biochimica et biophysica acta》1987,918(3):260-266
The role of purified plasma lipid transfer protein complexes in determining the particle size distribution of human plasma high density lipoproteins (HDL) was examined in vitro. Incubation of HDL2 or HDL3, isolated from normolipemic subjects with very low density lipoproteins (VLDL) or VLDL-remnants and lipid transfer protein complex had little or no effect on HDL particle size. In contrast, HDL isolated from patients with hypertriglyceridemia, designated HDL3D, showed speciation of particle size distribution when incubated with VLDL-remnants and the transfer protein. Incubation of HDL3D with VLDL-remnants and lipid transfer complex resulted in the production of two particles of radius 4.3 and 3.7 nm; incubation with VLDL or in the absence of the transfer protein did not result in a redistribution of particle size. We suggest that the action of lipid transfer protein complex on triacylglycerol-rich lipoprotein remnants and HDL accounts for the low levels of HDL-cholesterol observed in subjects with severe hypertriglyceridemia. 相似文献
10.
Human glycolipid transfer protein: probing conformation using fluorescence spectroscopy 总被引:1,自引:0,他引:1
Li XM Malakhova ML Lin X Pike HM Chung T Molotkovsky JG Brown RE 《Biochemistry》2004,43(31):10285-10294
Glycolipid transfer protein (GLTP) is a soluble 24 kDa protein that selectively accelerates the intermembrane transfer of glycolipids in vitro. Little is known about the GLTP structure and dynamics. Here, we report the cloning of human GLTP and characterize the environment of the three tryptophans (Trps) of the protein using fluorescence spectroscopy. Excitation at 295 nm yielded an emission maximum (lambda(max)) near 347 nm, indicating a relatively polar average environment for emitting Trps. Quenching with acrylamide at physiological ionic strength or with potassium iodide resulted in linear Stern-Volmer plots, suggesting accessibility of emitting Trps to soluble quenchers. Insights into reversible conformational changes accompanying changes in GLTP activity were provided by addition and rapid dilution of urea while monitoring changes in Trp or 1-anilinonaphthalene-8-sulfonic acid fluorescence. Incubation of GLTP with glycolipid liposomes caused a blue shift in the Trp emission maximum but diminished the fluorescence intensity. The blue-shifted emission maximum, centered near 335 nm, persisted after separation of glycolipid liposomes from GLTP, consistent with formation of a GLTP-glycolipid complex at a glycolipid-liganding site containing Trp. The results provide the first insights into human GLTP structural dynamics by fluorescence spectroscopy, including global conformational changes that accompany GLTP folding into an active conformational state as well as more subtle conformational changes that play a role in GLTP-mediated transfer of glycolipids between membranes, and establish a foundation for future studies of membrane rafts using GLTP. 相似文献
11.
Next to the protein-based machineries composed of small G-proteins, coat complexes, SNAREs and tethering factors, the lipid-based machineries are emerging as important players in membrane trafficking. As a component of these machineries, lipid transfer proteins have recently attracted the attention of cell biologists for their involvement in trafficking along different segments of the secretory pathway. Among these, the four-phosphate adaptor protein 2 (FAPP2) was discovered as a protein that localizes dynamically with the trans-Golgi network and regulates the transport of proteins from the Golgi complex to the cell surface. Later studies have highlighted a role for FAPP2 as lipid transfer protein involved in glycosphingolipid metabolism at the Golgi complex. Here we discuss the available evidence on the function of FAPP2 in both membrane trafficking and lipid metabolism and propose a mechanism of action of FAPP2 that integrates its activities in membrane trafficking and in lipid transfer. This article is part of a Special Issue entitled Lipids and Vesicular Transport. 相似文献
12.
Progressive truncations C terminal to the membrane-spanning domain of simian immunodeficiency virus Env reduce fusogenicity and increase concentration dependence of Env for fusion
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The simian immunodeficiency virus (SIV) transmembrane (TM) protein, gp41, has multiple functions, which include anchoring the glycoprotein complex in the lipid envelope of the virus and mediating fusion of the virus and host cell membranes. Recently, a series of mutants of the SIVmac239 TM protein that have truncations at the carboxyl terminus of the membrane-spanning domain (MSD) have been characterized (J. T. West, P. Johnston, S. R. Dubay, and E. Hunter, J. Virol. 75:9601-9612, 2001). These mutants retained membrane anchorage but demonstrated reduced fusogenicity and infectivity as the MSD length was shortened. We have established a novel three-color fluorescence assay, which allows qualitative confocal and quantitative flow cytometric analyses, to further characterize the nature of the fusion defect in five of the MSD mutants: TM185, TM186, TM187, TM188, and TM189. Our analysis showed that each mutant could mediate complete lipid and aqueous dye transfer at early time points after effector and target cell mixing. No hemifusion with only lipid dye flux was detected. However, another intermediate fusion stage, which appears to involve small-fusion-pore formation that allowed small aqueous dye transfer but prevented the exchange of large cytoplasmic components, was identified infrequently in mutant-Env-expressing cell and target cell mixtures. Quantitative flow cytometric analysis of these mutants demonstrated that the TM187, TM188, and TM189 mutants were significantly more fusogenic than TM185 and TM186 but remained significantly impaired compared to the wild type. Moreover, fusion efficiency showed an increased dependence on the expression level of glycoproteins, suggesting that, for these mutants, formation of an active fusion complex was an increasingly stochastic event. 相似文献
13.
The tryptophan intrinsic fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers was examined at different temperatures. Absorption and emission maxima occur at 277 and 332 nm, irrespective of temperature or lipid:protein ratio even if there are indications (from fluorescence quenching) of protein conformational changes as a function of lipid:protein ratio. Low values of Trp fluorescence quantum yield in complex III (0.008-0.010) are probably due to the neighborhood of the heme groups. The temperature-dependent decrease of fluorescence intensity is nonlinear; the corresponding Arrhenius plots show "breaks" or discontinuities that could be interpreted as thermally dependent changes in protein conformation. However, no temperature-dependent changes in fluorescence quenching have been observed that may be related to protein conformational changes. In addition, Arrhenius plots of the fluorescence intensity of simple molecules, such as Trp or 1-anilino-8-naphthalene sulfonate in the presence of aqueous phospholipid dispersions, also show breaks in the same temperature range. Stern-Volmer plots of acrylamide and iodide quenching were also nonlinear, indicating large differences in quenching constants for the various tryptophanyl residues. The quenching results also suggest that, at high lipid:protein ratios, the microviscosity of the protein matrix is higher than that in lipid-poor systems. Comparison of quenching efficiencies of iodide and acrylamide suggest that no significant fraction of the fluorophores occurs in the neighborhood of charged residues. 相似文献
14.
The phosphatidylinositol transfer protein isolated from brain, liver, heart and platelets was found to be present in two subforms which could be distinguished on the basis of the isoelectric points. In this study we have demonstrated that the two subforms isolated from bovine brain are due to the presence of either phosphatidylinositol or phosphatidylcholine in the lipid binding site of the protein. The transfer protein accommodates one phosphatidylinositol molecule in the binding site. The binding site for the sn-2 fatty acyl chain was investigated by incorporating in the transfer protein either phosphatidylinositol or phosphatidylcholine carrying a parinaroyl-chain attached at the sn-2 position. Time-resolved fluorescence spectroscopy revealed that the sn-2 fatty acyl chains for both phospholipids in the lipid-protein complex were completely immobilized (i.e., rotational correlation times of 17.4 ns for phosphatidylcholine and 16.3 ns for phosphatidylinositol). The similarity in correlation times suggests that the sn-2 fatty acyl chains of both phospholipids are accommodated in the same hydrophobic binding site of the protein. 相似文献
15.
Physical characteristics of binding of protein kinase C with sphingomyelin/cholesterol lipid bilayers were analysed using three complementary approaches: acrylodan fluorescence, fluorescence energy transfer and quenching of tryptophan fluorescence. It was demonstrated that sphingomyelin/cholesterol lipid membranes were available for protein kinase C binding. The intensity of the binding was dependent on the sphingomyelin content. The results of quenching of intrinsic tryptophan fluorescence showed that the enzyme molecule penetrated the sphingomyelin/cholesterol lipid bilayer to the C-16 position of labeled fatty acid probes. Our results also showed sphingomyelin itself restrains protein kinase C activity. A possible explanation for our results is that caveolae function as signaling storage devices. 相似文献
16.
Kinetics of fluorescent-labeled phosphatidylcholine transfer between nonspecific lipid transfer protein and phospholipid vesicles 总被引:2,自引:0,他引:2
J W Nichols 《Biochemistry》1988,27(6):1889-1896
Recently, rat liver nonspecific lipid transfer protein (nsLTP) was shown to form a fluorescent complex when allowed to equilibrate with self-quenching vesicles prepared from the fluorescent phospholipid 1-palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4- yl)amino]dodecanoyl]phosphatidylcholine (P-C12-NBD-PC) [Nichols, J. W. (1987) J. Biol. Chem. 262, 14172-14177]. Investigation of the mechanism of complex formation was continued by studying the kinetics of transfer of P-C12-NBD-PC between nsLTP and phospholipid vesicles using a transfer assay based on resonance energy transfer between P-C12-NBD-PC and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine. The principles of mass action kinetics (which predict initial lipid transfer rates as a function of protein and vesicle concentration) were used to derive equations for two distinct mechanisms: lipid transfer by the diffusion of monomers through the aqueous phase and lipid transfer during nsLTP-membrane collisions. The results of these kinetics studies indicated that the model for neither mechanism alone adequately predicted the initial rates of formation and dissolution of the P-C12-NBD-PC-nsLTP complex. The initial rate kinetics for both processes were predicted best by a model in which monomer diffusion and collision-dependent transfer occur simultaneously. These data support the hypothesis that the phospholipid-nsLTP complex functions as an intermediate in the transfer of phospholipids between membranes. 相似文献
17.
Fluorescence energy transfer in two dimensions. A numeric solution for random and nonrandom distributions. 总被引:7,自引:4,他引:3
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A method of Monte Carlo calculations has been applied to the problem of fluorescence energy transfer in two dimensions in order to provide a quantitative measure of the effects of nonideal mixing of lipid and protein molecules on the quenching profiles of membrane systems. These numerical techniques permit the formulation of a detailed set of equations that describes in a precise manner the quenching and depolarization properties of planar donor-acceptor distributions as a function of specific spectroscopic and organizational parameters. Because of the exact nature of the present numeric method, these results are used to evaluate critically the validity of previous approximate treatments existing in the literature. This method is also used to examine the effects of excluded volume interactions and distinct lattice structures on the expected transfer efficiencies. As a specific application, representative quenching profiles for protein-lipid mixtures, in which donor groups are covalently linked to the protein molecules and acceptor species are randomly distributed within lipid domains, have been obtained. It is found that the existence of phase-separated protein domains gives rise to a shielding effect that significantly decreases the transfer efficiencies with respect to those expected for an ideal distribution of protein molecules. The results from the present numerical study indicate that the experimental application of fluorescence energy transfer measurements in multicomponent membrane systems can be used to obtain organizational parameters that accurately reflect the lateral distribution of protein and lipid molecules within the bilayer membrane. 相似文献
18.
The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface. 相似文献
19.
Phosphoinositides are important signal transduction intermediates in cell growth, survival, and motility. We have invented a fluorescence sensor for polyphosphorylated phosphoinositides based on a peptide derived from the Listeria protein ActA that undergoes a random coil to helix transition upon lipid binding. The sensor, termed CAY, is a fusion protein of cyan and yellow fluorescent proteins flanking the peptide at its N- and C-termini, respectively. CAY displays fluorescence resonance energy transfer in vitro in the absence of phosphorylated phosphoinositides, and this energy transfer is lost upon interaction with these phospholipids. These results demonstrate that a short peptide undergoing a coil to helix transition can be sufficient for the engineering of a FRET-based biosensor. CAY is predominantly localized to the cytoplasm in fibroblasts expressing the sensor but shows loss of fluorescence resonance energy transfer in regions of active actin dynamics such as ruffles that have previously been demonstrated to contain high levels of phosphoinositides. 相似文献
20.
The interaction of lipoprotein lipase (LpL) and its activator protein, apolipoprotein C-II (apoC-II), with a nonhydrolyzable phosphatidylcholine, 1,2-ditetradecyl-rac-glycero-3-phosphocholine (C14-ether-PC), was studied by fluorescence spectroscopy. A complex of 320 molecules of C14-ether-PC per LpL was isolated by density gradient ultracentrifugation in KBr. The intrinsic tryptophan fluorescence emission spectrum of LpL was shifted from 336 nm in the absence of lipid to 330 nm in the LpL-lipid complex; the shift was associated with a 40% increase in fluorescence intensity. Addition of C14-ether-PC vesicles to apoC-II caused a 2.5-fold increase in intrinsic tryptophan fluorescence and a shift in emission maximum from 340 to 317 nm. LpL and apoC-II/C14-ether-PC stoichiometries and binding constants were determined by measuring the increase in the intrinsic tryptophan fluorescence as a function of lipid and protein concentrations; for LpL the rate and magnitude of the fluorescence increases were relatively independent of temperature in the range 4-37 degrees C. A stoichiometry of 270 PC per LpL for the LpL-lipid complex compares favorably with the value obtained in the isolated complex. The dissociation constant (Kd) of the complex is 4.3 X 10(-8) M. For apoC-II, the stoichiometry of the complex is 18 PC per apoprotein, and the Kd is 3.0 X 10(-6) M. These data suggest that LpL binds more strongly than apoC-II to phosphatidylcholine interfaces. 相似文献