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1.
The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly 总被引:4,自引:4,他引:0
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《The Journal of cell biology》1993,120(4):909-922
Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F- actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization. 相似文献
2.
C Kerkhoff B Trümbach L Gehring K Habben G Schmitz V Kaever 《European journal of biochemistry》2000,267(21):6339-6345
In the present study, we defined experimental conditions that allowed the extraction of the integral membrane protein lysophospholipid:acyl-CoA acyltransferase (LAT, EC 2.3.1.23) from membranes while maintaining the full enzyme activity using the nonionic detergent n-octyl glucopyranoside (OGP) and solutions of high ionic strength. We found that the optimal OGP concentration depended on the ionic strength of the solubilization buffer. Fluorescence measurements with 1,6-diphenyl-1,3,5-hexatriene indicated that the critical micellar concentration (CMC) of OGP decreased with increasing salt concentrations. Analogous studies revealed that the zwitterionic detergent Chaps was ineffective in extracting LAT from membranes in the absence of salt, whereas its solubilization efficiency increased with increasing salt concentrations. Detailed lipid analysis of the different protein/lipid/detergent mixed micelles showed that the protein/lipid/OGP mixed micelles were relatively enriched with sphingomyelin (SPM) compared to protein/lipid/Chaps mixed micelles, indicating that the differences in the solubilization efficiency may be due to the ability to extract more SPM from membranes. When the protein/lipid/OGP mixed micelles were dissociated into protein/detergent and lipid/detergent complexes by the addition of increasing Chaps concentrations, one-tenth of the LAT enzyme activity was preserved making the enzyme accessible to protein purification. Analysis by native PAGE revealed that in the presence of excess Chaps a high molecular mass protein complex migrated into the gel which could be photolabeled by 125I-labelled-18-(4'-azido-2'-hydroxybenzoylamino)-oleyl-CoA. This fatty acid analogue has been shown to be a competitive inhibitor of LAT enzyme activity in the dark, and an irreversible inhibitor after photolysis. Therefore, this protein complex is assumed to contain the LAT enzyme. 相似文献
3.
Paul G. Crichton Marilyn Harding Jonathan J. Ruprecht Yang Lee Edmund R. S. Kunji 《The Journal of biological chemistry》2013,288(30):22163-22173
Blue native gel electrophoresis is a popular method for the determination of the oligomeric state of membrane proteins. Studies using this technique have reported that mitochondrial carriers are dimeric (composed of two ∼32-kDa monomers) and, in some cases, can form physiologically relevant associations with other proteins. Here, we have scrutinized the behavior of the yeast mitochondrial ADP/ATP carrier AAC3 in blue native gels. We find that the apparent mass of AAC3 varies in a detergent- and lipid-dependent manner (from ∼60 to ∼130 kDa) that is not related to changes in the oligomeric state of the protein, but reflects differences in the associated detergent-lipid micelle and Coomassie Blue G-250 used in this technique. Higher oligomeric state species are only observed under less favorable solubilization conditions, consistent with aggregation of the protein. Calibration with an artificial covalent AAC3 dimer indicates that the mass observed for solubilized AAC3 and other mitochondrial carriers corresponds to a monomer. Size exclusion chromatography of purified AAC3 in dodecyl maltoside under blue native gel-like conditions shows that the mass of the monomer is ∼120 kDa, but appears smaller on gels (∼60 kDa) due to the unusually high amount of bound negatively charged dye, which increases the electrophoretic mobility of the protein-detergent-dye micelle complex. Our results show that bound lipid, detergent, and Coomassie stain alter the behavior of mitochondrial carriers on gels, which is likely to be true for other small membrane proteins where the associated lipid-detergent micelle is large when compared with the mass of the protein. 相似文献
4.
Heymann JA Sarker R Hirai T Shi D Milne JL Maloney PC Subramaniam S 《The EMBO journal》2001,20(16):4408-4413
The major facilitator superfamily (MFS) represents the largest collection of evolutionarily related members within the class of membrane 'carrier' proteins. OxlT, a representative example of the MFS, is an oxalate-transporting membrane protein in Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional crystals of OxlT, we have determined the projection structure of this membrane transporter. The projection map at 6 A resolution indicates the presence of 12 transmembrane helices in each monomer of OxlT, with one set of six helices related to the other set by an approximate internal two-fold axis. The projection map reveals the existence of a central cavity, which we propose to be part of the pathway of oxalate transport. By combining information from the projection map with related biochemical data, we present probable models for the architectural arrangement of transmembrane helices in this protein superfamily. 相似文献
5.
OxlT, a secondary carrier found in Oxalobacter formigenes, mediates the exchange of divalent oxalate and monovalent formate. Because OxlT has an unusually high turnover number (greater than or equal to 1000/s), and because formate, one its substrates, shows high passive membrane permeability as formic acid, it has been difficult to obtain information on protein-substrate interactions using traditional methods in membrane biology. For this reason, we devised a new way to measure substrate dissociation constants. Detergent-solubilized material was exposed to inactivating temperatures in the absence or presence of OxlT substrates, and periodic reconstitution was used to monitor the kinetics of thermal decay. The data were consistent with a simple scheme in which only unliganded OxlT was temperature-sensitive; this premise, along with the assumption of equilibrium between liganded and unliganded species, allowed calculation of substrate dissociation constants for oxalate (18 +/- 3 microM), malonate (1.2 +/- 0.2 mM), and formate (3.1 +/- 0.6 mM). Further analysis revealed that substrate binding energy contributed at least 3.5 kcal/mol to stabilization of solubilized OxlT. Accordingly, we suggest that substrate binding energy is directly involved in driving protein structure reorganization during membrane transport. This new approach to analyzing protein-substrate interactions may have wider application in the study of membrane carriers. 相似文献
6.
Escherichia coli diacylglycerol kinase (DAGK) is a homotrimeric helical integral membrane protein in which a number of single-site mutations to cysteine are known to promote misfolding. Here, effects of other amino acid replacements have been explored using a folding assay based on the dilution of acidic urea/DAGK stock solutions into detergent/lipid mixed micelles. DAGK with an I110P or I110R mutation in the third transmembrane helix could not be purified because its expression was toxic to the E. coli host, most likely because of severe folding defects. Other mutations at Ile110 enhanced irreversible misfolding to varying degrees that generally correlated both with the polarity of the inserted amino acid and with the degree of protein destabilization. However, the I110W mutant was an exception in that it was highly misfolding prone while at the same time being more stable than the wild-type protein. This contrasts with I110Y, which also exhibited enhanced stability but folded with an efficiency similar to that of the wild type. For most mutants, the critical step leading to irreversible misfolding occurred for monomeric DAGK prior to trimerization and independent of association with mixed micelles. Misfolding of DAGK evidently involves the formation of incorrect monomer tertiary structure. Mutations appear to enhance misfolding by disfavoring the formation of correct structure rather than by directly stabilizing the misfolded state. Finally, when urea-solubilized DAGK was diluted into detergent/lipid-free buffer, it retained a significant degree of folding competency over a period of minutes. This property may be relevant to membrane protein folding in cells under conditions where the usual machinery associated with membrane integration is saturated, dysregulated, or dysfunctional. 相似文献
7.
The use of neutron scattering in studying the organisation of detergents in pure micelles, in protein/detergent mixed micelles and in crystals of membrane proteins, is reviewed. Small angle scattering has been used to study the size, shape and composition of pure and mixed protein/detergent micelles as well as the effects of adding small amphiphiles. The technique of contrast variation applied to single crystals is described and its application to the determination of the organization of detergent in single crystals of membrane proteins is discussed. A better understanding of protein/detergent interactions should help in producing crystals of membrane proteins more easily as well as clues to the nature of protein/lipid interactions in vivo. 相似文献
8.
Chaowei Shi Fang Han Ying Xiong Changlin Tian 《Protein expression and purification》2009,68(2):221-225
In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC–MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies. 相似文献
9.
The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein. 相似文献
10.
Z S Ruan V Anantharam I T Crawford S V Ambudkar S Y Rhee M J Allison P C Maloney 《The Journal of biological chemistry》1992,267(15):10537-10543
We had proposed earlier that the anaerobe Oxalobacter formigenes sustains a proton-motive force by exploiting a secondary carrier rather than a primary proton pump. In this view, a carrier protein would catalyze the exchange of extracellular oxalate, a divalent anion, and intracellular formate, the monovalent product of oxalate decarboxylation. Such an electrogenic exchange develops an internally negative membrane potential, and since the decarboxylation reaction consumes an internal proton, the combined activity of the carrier and the soluble decarboxylase would constitute an "indirect" proton pump with a stoichiometry of 1H+ per turnover. This model is now verified by identification and purification of OxlT, the protein responsible for the anion exchange reaction. Membranes of O. formigenes were solubilized at pH 7 with 1.25% octyl glucoside in 20 mM 3-(N-morpholino)propanesulfonic acid/K, in the presence of 0.4% Escherichia coli phospholipids and with 20% glucerol present as the osmolyte stabilant. Rapid methods for reconstitution were developed to monitor the distribution of OxlT during biochemical fractionation, allowing its purification by sequential anion and cation exchange chromatography. OxlT proved to be a single hydrophobic polypeptide, of 38 kDa mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a turnover number estimated as at least 1000/s. The properties of OxlT point to an indirect proton pump as the mechanism by which a proton-motive force arises in O. formigenes, and one may reasonably argue that indirect proton pumps take part in bacterial events such as acetogenesis, malolactate fermentation, and perhaps methanogenesis. 相似文献
11.
Hydrodynamic properties of colicin A. Existence of a high-affinity lipid-binding site and oligomerization at acid pH 总被引:3,自引:0,他引:3
D Cavard P Sauve F Heitz F Pattus C Martinez R Dijkman C Lazdunski 《European journal of biochemistry》1988,172(2):507-512
The hydrodynamic properties of colicin A have been studied. The molecular mass of colicin A was determined from sedimentation equilibrium centrifugation to be 63 +/- 1.2 kDa, in agreement with that determined from the primary amino acid sequence [Morlon et al. (1983) J. Mol. Biol. 110, 271-289]. The sedimentation coefficient has been analyzed over a wide range of ionic strength (NaCl 0.06-0.56 M) and pH (8-4) and was found to remain almost constant. However, below pH 5 an oligomerization of colicin A to tetramers occurred. The frictional coefficient value indicated that the shape of the colicin A monomer was very asymmetric. Analysis of the pH dependence of circular dichroism of colicin A and of its COOH-terminal domain indicated that a sharp transition occurred between pH 4 and 3. This transition was very much reduced for the COOH-terminal domain in the presence of a non-ionic detergent. The presence of a lipid-binding site in colicin A at neutral pH was demonstrated both by hydrodynamic studies with micelles of n-hexadecanoyl and n-octadecanoylphosphocholine and by differential sensitivity to a proteolytic enzyme in the presence or absence of detergent micelles. About 75 molecules of lipid were bound under these conditions suggesting that colicin A was bound to lipid micelles. In contrast, at acid pH, in the presence of an excess of lipid the tetramer was dissociated into monomers complexed to 20-30 lipid molecules, indicating the exposure of a high-affinity lipid-binding site. 相似文献
12.
Membrane proteins, lipids and detergents: not just a soap opera 总被引:1,自引:0,他引:1
Studying membrane proteins represents a major challenge in protein biochemistry, with one of the major difficulties being the problems encountered when working outside the natural lipid environment. In vitro studies such as crystallization are reliant on the successful solubilization or reconstitution of membrane proteins, which generally involves the careful selection of solubilizing detergents and mixed lipid/detergent systems. This review will concentrate on the methods currently available for efficient reconstitution and solubilization of membrane proteins through the use of detergent micelles, mixed lipid/detergent micelles and bicelles or liposomes. We focus on the relevant molecular properties of the detergents and lipids that aid understanding of these processes. A significant barrier to membrane protein research is retaining the stability and function of the protein during solubilization, reconstitution and crystallization. We highlight some of the lessons learnt from studies of membrane protein folding in vitro and give an overview of the role that lipids can play in stabilizing the proteins. 相似文献
13.
This article examines the feasibility of using ultrafiltration to separate the monomer of the monoclonal antibody alemtuzumab (Campath or Campath-1H) from a mixture of dimer and higher-order oligomers (collectively called "dimers" here). Using parameter scanning ultrafiltration, we initially assessed the suitability of the following membranes: 100 kDa and 300 kDa polyethersulfone (PES) membranes, and a 100 kDa polyvinylidene fluoride (PVDF) membrane. A detailed study was then carried out to examine the effects of operating conditions (such as solution pH, ionic strength, stirring speed, and permeate flux) on the separation of the monomer from the dimers using 300 kDa PES and 100 kDa PVDF membranes. Results of the experiments carried out in the carrier phase ultrafiltration (CPUF) mode indicate that the size-based protein-protein separation critically depends on the membrane used as well as the system hydrodynamics. The separation of the monoclonal antibody monomer and dimers using 100 kDa PVDF membranes in the diafiltration mode was also examined. Experimental results demonstrate that under suitable conditions, it is feasible to obtain the alemtuzumab monomer with a purity of more than 93% and a yield of more than 85% (from a mixture of 75% monomer and 25% dimers, which is the typical composition obtained after affinity chromatography). Simulation study indicates that this could be further improved to a purity of more than 96% and a monomer yield of more than 96% by increasing the selectivity of separation or by employing a two-stage diafiltration process. 相似文献
14.
We constructed a single cysteine panel encompassing transmembrane helix two (TM2) of OxlT, the oxalate/formate antiporter of Oxalobacter formigenes. Among the 21 positions targeted, cysteine substitution identified one (phenylalanine 59) as essential to OxlT expression and three (glutamine 56, glutamine 66, and serine 69) as potentially critical to OxlT function. By probing membranes with a bulky hydrophilic probe (Oregon Green maleimide) we also located a central inaccessible core of at least eight residues in length, extending from leucine 61 to glycine 68. Functional assays based on reconstitution of crude detergent extracts showed that of single cysteine mutants within the TM2 core only the Q63C variant was substantially (> or =95%) inhibited by thiol-specific agents (carboxyethyl methanethiosulfonate and ethylsulfonate methanethiosulfonate). Subsequent analytical work using the purified Q63C protein showed that inhibition by ethylsulfonate methanethiosulfonate was blocked by substrate and that the concentration dependence of such substrate protection occurred with a binding constant of 0.16 mm oxalate, comparable with the Michaelis constant observed for oxalate transport (0.23 mm). These findings lead us to conclude that position 63 lies on the OxlT translocation pathway. Our conclusion is strengthened by the finding that position 63, along with most other positions relevant to TM2 function, is found on a helical face that can be cross-linked to the pathway-facing surface of TM11 (Fu, D., Sarker, R. I., Bolton, E., and Maloney, P. C. (2001) J. Biol. Chem. 276, 8753-8760). 相似文献
15.
alpha-Synuclein (alphaS) is a presynaptic terminal protein that is believed to play an important role in the pathogenesis of Parkinson's disease (PD). We have used NMR spectroscopy to characterize the conformational properties of alphaS in solution as a free monomer and when bound to lipid vesicles and lipid-mimetic detergent micelles. Free wild-type alphaS is largely unfolded in solution, but exhibits a region with a preference for helical conformations that may be important in the aggregation of alphaS into fibrils. The N-terminal region of alphaS binds to synthetic lipid vesicles and detergent micelles in vitro and adopts a highly helical conformation, consistent with predictions based on sequence analysis. The C-terminal part of the protein does not associate with either vesicles or micelles, remaining free and unfolded. These results suggest that one function of alphaS may be to tether as of yet unidentified partners to lipid surfaces via interactions with its C-terminal tail. 相似文献
16.
《Journal of molecular biology》2021,433(8):166886
CLC-ec1 is a Cl−/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mol in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4–5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1. 相似文献
17.
Shigella dysenteriae, like many bacterial pathogens, has evolved outer membrane receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the outer membrane receptor ShuA of S. dysenteriae. Purification of the outer membrane receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1:1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent outer membrane receptors. 相似文献
18.
Cholesterol and its hemisuccinate and sulfate derivatives are widely used in studies of purified membrane proteins but are difficult to solubilize in aqueous solution, even in the presence of detergent micelles. Other cholesterol derivatives do not form conventional micelles and lead to viscous solutions. To address these problems, a cholesterol-based detergent, CHOBIMALT, has been synthesized and characterized. At concentrations above 3?4 μM, CHOBIMALT forms micelles without the need for elevated temperatures or sonic disruption. Diffusion and fluorescence measurements indicated that CHOBIMALT micelles are large (210±30 kDa). The ability to solubilize a functional membrane protein was explored using a G-protein coupled receptor, the human kappa opioid receptor type 1 (hKOR1). While CHOBIMALT alone was not found to be effective as a surfactant for membrane extraction, when added to classical detergent micelles CHOBIMALT was observed to dramatically enhance the thermal stability of solubilized hKOR1. 相似文献
19.
Membrane proteins that belong to the major facilitator superfamily (MFS) are found in organisms across the evolutionary spectrum and mediate the transport of a variety of substrates ranging from small metabolites to neurotransmitters. The oxalate transporter (OxlT) is a representative MFS protein, and exchanges formate for oxalate across the cytoplasmic membrane of the organism Oxalobacter formigenes. Here, we present a structural model for the protein conformational changes that occur during oxalate transport by combining a three-dimensional map of the oxalate-bound, "closed" state of OxlT at 6.5 A determined by cryo-electron microscopy with a model of the "open" state of OxlT based on the atomic structures of the related transporters, glycerol-3-phosphate transporter (GlpT) and lactose permease (LacY). We demonstrate that the principal structural change associated with substrate transport is a concerted rocking movement of the two structurally similar halves of the protein relative to each other. Our structural model places two positively charged residues, Arg-272 and Lys-355 in the central cavity, suggesting that electrostatic interactions between these residues and the oxalate anion is a key step in generating the conformational change between the open and closed states of the transporter. 相似文献
20.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels. 相似文献