Effect of detergent on protein structure. Action of detergents on secondary and oligomeric structures of band 3 from bovine erythrocyte membranes

With special interest in the mode of action of zwitterionic detergents on proteins, a variety of detergents were examined for their ability to disrupt the secondary and quaternary structures of an anion transport protein, band 3, and its cytoplasmic 38 kDa fragment from bovine erythrocyte membranes and for their effect on the binding of an anion transport inhibitor to band 3. Nonionic detergents and Chaps also acted as a nondenaturant in these instances, as well accepted for other proteins. Though deoxycholate and cholate inhibited the binding of an anion transport inhibitor to band 3, these detergents did not show any effect on the native structure of band 3. Zwitterionic detergents (Zwittergent 3-10, Zwittergent 3-12 and N, N-dimethyl-N-dodecyl glycine) were suggested to denature the water-soluble 38 kDa fragment at concentrations above the critical micelle concentration, but to be weak in disrupting interacting forces between hydrophobic membrane-bound domains of band 3. The results indicated that these zwitterionic detergents are similar in the mode of denaturing action to dodecyltrimethylammonium bromide rather than sodium dodecyl sulfate.     

Direct immunological identification of proteins following electrophoretic separation in polyacrylamide micro-gradient gels and the effect of detergents on immunoprecipitation (author's transl)]

Methods for direct immunological identification of single protein components after fractionation of a protein mixture in microgels are described. Protein mixtures were separated with high resolution in polyacrylamide microgradient gels and transferred after electrophoresis into agarose layers containing suitable antisera. Monospecific as well as polyvalent antisera were used. The formation of immunoprecipitates could be observed within approx. 1 h. Immunoprecipitates are also formed in the presence of sodium dodecylsulfate or other detergents, thus allowing immunoreactions to be performed with water-insoluble proteins. Staining of the proteins in the gels did not completely inhibit the immunoreaction, while dansylation of proteins had no effect. The influence of different detergents e.g. sodium dodecylsulfate, Triton X-100, Brij 99, np-40 and urea, as well as different reducing agents e.g. mercaptoethanol, dithiothreitol, thioglycolic acid, on two-dimensional microdiffusion was also studied. When suitable concentrations of these compounds were used, the formation of immunocomplexes was observed within approx. 1 h. This technique can also be applied to immunoreactions with water-insoluble proteins dissolved in detergents.     

Solubilization and reconstitution of membrane proteins of Escherichia coli using alkanoyl-N-methylglucamides

Alkanoyl-N-methylglucamides, nonionic detergents, were utilized to solubilize membrane proteins of Escherichia coli and were used to reconstitute them into liposomes. First, critical micelle concentrations (CMC) of nonanoyl-N-methylglucamide and decanoyl-N-methylglucamide were determined to be 25 mM and 7 mM, respectively, by photometric assay. Then solubilization and reconstitution of the melibiose transport carrier were performed using these detergents at concentrations above the CMC. Melibiose counterflow activity was observed with the proteoliposomes reconstituted from the extracted proteins and phospholipids. The proton-translocating ATPase complex (F1-F0) was also solubilized with these detergents. These results indicate that nonanoyl- and decanoyl-N-methylglucamide are useful detergents for solubilization and reconstitution of membrane proteins.     

Comparative extraction of erythrocyte EDTA-membrane proteins by some ionic and non-ionic detergents

In order to examine whether it would be possible to obtain, by a simple extraction procedure from EDTA-erythrocyte-membranes, a partially purified preparation of the "band 3 zone" proteins, we have tested four solubilizing agents of common use. Detergents, both ionic (DOC and SDS) and non ionic (Tween 80 and Triton X-100), were not able, in our experimental conditions, to completely solubilize erythrocyte fragmented membranes which had previously been washed in EDTA-buffers. However, they were able to solubilize some of the membrane proteins, which could then be separated by SDS-PGE. The PGE densitometric profiles reported in this communication indicate that the protein mixture extracted by the ionic detergents DOC and SDS qualitatively reflects the protein composition of the membranes. Among the non ionic detergents, on the other hand, Triton X-100 appeared to be able to extract mainly one band (most probably the band 3 zone), while Tween 80 did not apparently extract any of the membrane proteins. Detergent concentrations, medium composition and experimental procedures are described in detail.     

Optimization of detergents in solubilization and reconstitution of Aquaporin Z: A structural approach

BackgroundThe exceptional capacities of aquaporins in terms of water permeation and selectivity have made them an interesting system for membrane applications. Despite the multiple attempts for immobilizing the aquaporins over a porous substrate, there is a lack of studies related to the purification and reconstitution steps, principally associated with the use of detergents in solubilization and destabilization steps. This study analyzed the effect of detergents in Aquaporin Z solubilization, considering the purity and structural homogeneity of the protein.MethodsThe extraction process was optimized by the addition of detergent at the sonication step, which enabled the omission of the ultracentrifugation and resuspension steps. Two detergents, Triton X-100, and octyl-glucoside were also evaluated. Destabilization mediated by detergents was used as reconstitution method. Saturation and solubilization points were defined by detergent concentration and both, liposomes and proteoliposomes, were analyzed by size distribution and permeability assays. Detergent removal with Bio-beads was also analyzed.ResultsOctyl glucoside ensures structural stability and homogeneity of Aquaporin Z. However, high concentrations of detergents induce the presence of defects in proteoliposomes. While saturated liposomes create homogeneous and functional structures, solubilized liposomes get affected by a reassembly process, creating vesicle defects with anomalous permeability profiles.ConclusionsDetergent concentration affects the structural conformation of proteoliposomes in the reconstitution process.General significanceSince the destabilization process is dependent on vesicle, detergent, and buffer composition, optimization of this process should be mandatory for further studies. All these considerations will allow achieving the potential of Aquaporins and any other integral membrane protein in their applications for industrial purposes.     

Detection of the associated state of membrane proteins by polyacrylamide gradient gel electrophoresis with non-denaturing detergents Application to band 3 protein from erythrocyte membranes

Polyacrylamide gradient gel electrophoresis was carried out in micellar solutions of various detergents which differ in degree of potency to denature proteins. From the application of this method to band 3 protein from erythrocyte membranes, it was suggested that the procedure was useful in studying the molecular state of membrane proteins.The electrophoretic behaviors of human and bovine band 3 protein did not show any species specificity in either a denature state and a state resembling the native state. As well as in nonionic detergent solutions, the dimeric and tetrameric structures of bovine band 3 protein were preserved in sodium deoxycholate solution, in which protein complexes maintained in nonionic detergent solutions are frequently dissociated. Even in dodecyltrimethylammonium bromide solution, which is a denaturant for water-soluble proteins, part of the band 3 protein was still present as the oligomer. The results suggest that the oligomeric form of band 3 protein is the stable structure and that the dimer and tetramer possibly coexist in membranes.     

Ultrastructure of human erythrocyte GLUT1

This study was undertaken to examine GLUT1 quaternary structure. Independent but complementary methodologies were used to investigate the influence of membrane-solubilizing detergents on GLUT1/lipid/detergent micelle hydrodynamic radii. Hydrodynamic size analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze-fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. GLUT1 forms a multimeric complex in octyl glucoside that dissociates upon addition of reductant. Some detergents (e.g., CHAPS and dodecyl maltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). These complexes do not reassemble as larger oligomers when dissociating detergents are subsequently replaced with nondissociating detergents such as octyl glucoside or cholic acid. When dissociating detergents are replaced with lipids, the resulting proteoliposomes catalyze protein-mediated sugar transport, and the subsequent addition of solubilizing, nondissociating detergents generates higher (tetrameric) GLUT1 aggregation states. These findings suggest that some detergents stabilize while others destabilize GLUT1 quaternary structure. GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer.     

Reconstitution of a partially purified Na+-dependent D-glucose transport system from rat jejunal brush border membranes

The Na+-dependent D-glucose transport system of rat jejunal brush border membranes was partially purified and reconstituted into functional proteoliposomes. Brush border membrane vesciles isolated from villous cells were first extracted with 0.3% cholate to remove extrinsic proteins and the insoluble residual pellet was reextracted with 1.2% cholate. The 1.2% cholate-extracted soluble fraction was then further purified by hydroxylapatite and Concanavalin A affinity chromatography in tandem. When the HLP-unadsorbed-ConA-unadsorbed fraction was reconstituted into proteoliposomes, it showed a characteristic Na+-coupled, phlorizin inhibitable, D-glucose transport activity that was 3 fold higher than that of the reconstituted proteoliposomes of the 1.2% cholate-extracted fraction. This partially purified fraction also displayed the simplest polypeptide composition pattern among all the membrane fractions analysed in SDS-polyacrylamide gels.     

Improved technique for reconstituting incredibly high and soluble amounts of tetrameric K⁺ channel in natural membranes

The reconstitution of large amounts of integral proteins into lipid vesicles is largely prompted by the complexity of most biological membranes and protein stability. We optimized a particular system which maximized the incorporation efficiency of large soluble amounts of KcsA potassium channel in Escherichia coli membranes. The effects of two detergents, octylglucoside and 3-[(cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), on KcsA reconstitution were compared. Reconstitution efficiency was found to be incredibly high for CHAPS-treated proteoliposomes followed by dialysis at room temperature. This approach may allow more accurate investigation of integral membrane proteins in their natural membrane environment via biophysical or biochemical techniques.     

Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 2. Incorporation of the light-driven proton pump bacteriorhodopsin

A method has been developed for identifying the step in a detergent-mediated reconstitution procedure at which an integral membrane protein can be associated with phospholipids to give functional proteoliposomes. Large liposomes prepared by reverse-phase evaporation were treated with various amounts of the detergents Triton X-100, octyl glucoside, or sodium cholate as described in the preceding paper [Paternostre, M.-T., Roux, M., & Rigaud, J. L. (1988) Biochemistry (preceding paper in this issue)]. At each step of the solubilization process, we added bacteriorhodopsin, the light-driven proton pump from Halobacterium halobium. The protein-phospholipid detergent mixtures were then subjected to SM2 Bio-Beads treatments to remove the detergent, and the resulting vesicles were analyzed with respect to protein insertion and orientation in the membrane by freeze-fracture electron microscopy, sucrose density gradients, and proton pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With sodium cholate, proteoliposomes were formed only from ternary phospholipid-protein-detergent micelles. With octyl glucoside, besides proteoliposome formation from ternary mixed micelles, direct incorporation of bacteriorhodopsin into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes with optimal proton pumping activity. With Triton X-100, protein insertion into destabilized liposomes was also observed but involved a transfer of the protein initially present in phospholipid-Triton X-100-protein micelles into Triton X-100 saturated liposomes. Our results further demonstrated that protein orientation in the resulting proteoliposomes was critically dependent upon the mechanism by which the protein was incorporated.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

A differential dilatometer

To obviate the difficulties resulting from partial solubility of membrane proteins in detergents or from the use of noxious solvent mixtures containing phenol or chloral, a simple procedure was devised for acrylamide gel electrophoresis of membrane proteins in 13 m formic acid. Polyacrylamide gels are equilibrated in 13 m formic acid and used in the electrophoresis assembly with 13 m formic acid as the electrolyte. Particulate proteinaceous preparations are dissolved in trifluoroacetic acid or in 24 m formic acid containing glycine to increase the density and to facilitate the solubilization of the protein. Protein samples (10 to 100 μg) migrate as polycations.     

A new method for the preparation of band 3, the main integral protein of the human erythrocyte membrane

Band-3 protein from human erythrocyte membranes was isolated, without using detergents, by a two-step procedure: (1) The peripheral proteins were removed from the membrane by treatment with 10% acetic acid. (2) The remaining lipoprotein complex was solubilized in approximately 92% (v/v) acetic acid and then separated into its components by preparative zonal electrophoresis in a gradient made up of acetic acid, water and sucrose. Band 3 was recovered from the gradient at a yield of 60 - 70% and purity of about 95%. Approximately 25 mg of band 3 could be prepared in one run. The protein is soluble in aqueous solutions, even in the absence of organic solvents or detergents. In addition to band 3, the proteins stained by periodic acid/Schiff's reagent (the sialoglycoproteins) are also separated from the other proteins.     

Optimization of mass spectrometry-compatible surfactants for shotgun proteomics

An optimization and comparison of trypsin digestion strategies for peptide/protein identifications by microLC-MS/MS with or without MS compatible detergents in mixed organic-aqueous and aqueous systems was carried out in this study. We determine that adding MS-compatible detergents to proteolytic digestion protocols dramatically increases peptide and protein identifications in complex protein mixtures by shotgun proteomics. Protein solubilization and proteolytic efficiency are increased by including MS-compatible detergents in trypsin digestion buffers. A modified trypsin digestion protocol incorporating the MS compatible detergents consistently identifies over 300 proteins from 5 microg of pancreatic cell lysates and generates a greater number of peptide identifications than trypsin digestion with urea when using LC-MS/MS. Furthermore, over 700 proteins were identified by merging protein identifications from trypsin digestion with three different MS-compatible detergents. We also observe that the use of mixed aqueous and organic solvent systems can influence protein identifications in combinations with different MS-compatible detergents. Peptide mixtures generated from different MS-compatible detergents and buffer combinations show a significant difference in hydrophobicity. Our results show that protein digestion schemes incorporating MS-compatible detergents generate quantitative as well as qualitative changes in observed peptide identifications, which lead to increased protein identifications overall and potentially increased identification of low-abundance proteins.     

Electrofocusing of integral membrane proteins in mixtures of zwitterionic and nonionic detergents.

In an attempt to fractionate mouse liver cytochrome P-450 in its native state, electrofocusing systems were examined under conditions in which the surface net charge of solubilized proteins was preserved. A mixture of the zwitterionic detergent, SB₁₄, and the nonionic detergent, Triton X-100, appeared capable of completely solubilizing intergral membrane proteins. Since charge properties were not altered, it was possible, for the first time, to focus basic membrane proteins in such detergent mixtures. The pH gradients (pI range 7–11) formed in the presence of these detergents were sufficiently stable to allow electrofocusing to the steady state of the solubilized membrane proteins. By the criterion of patter constancy, these conditions were achieved within 15 h, 0–4°C, at 200 V in 6-cm gels of 5% T/15% C_Bis with 0.1 n H₂SO₄ and 0.1 n KOH as anolyte and catholyte, respectively. It was expected that the native state of solubilized proteins could be maintained in such systems. Cytochrome P-450 proved to be denatured, however, by concentrations of these detergents required for complete solubilization of mouse liver endoplasmic reticulum.     

Glucose and nucleoside transporters of human erythrocytes: effects of detergents on immunoadsorption of a membrane protein to its monoclonal antibody.

Immunoadsorption of membrane proteins solubilized in detergents has been used widely for identification, purification and quantitation of transporters and receptors. In an effort to separate the glucose and nucleoside nucleoside transporters of human erythrocytes (GT and NT, respectively) that copurify in a membrane protein fraction band 4.5, we examined in the present study the effects of seven different detergents on the immunoadsorption of GT to its monoclonal antibody, 65D4 (Craik, et al. (1988) Biochem. Cell Biol. 66, 839-852). The following results were obtained. (1) The maximum extent of the immunoadsorption of GT by 65D4 varied between 52 to 98% in different detergents. For non-ionic detergents, there was an apparent inverse correlation between the maximum immunoreactivity of GT and the aggregation number or micellar size of detergents. (2) The immunoprecipitate of GT by 65D4 was contaminated with nucleoside transporter to an extent that varied from 2 to 35 mol% in different detergents. There is an inverse correlation between the extent of the contamination and the detergent aggregation number. However, this contamination was quantitatively accounted for by a time-dependent, non-specific aggregation of NT with GT in detergents. (3) A high degree of purification of NT in band 4.5 by immunoadsorptive removal of GT with 65D4 was achieved in C12E8 as predicted by the observed low NT-GT aggregation and the relatively high epitope-accessibility of GT in this detergent. Based on these findings, we conclude that certain detergents can reduce the immunoreactivity of membrane proteins significantly by modulating epitope accessibility, and may also produce a false immuno-cross-reactivity by inducing nonspecific protein aggregation.     

High Yield Elution of Proteins from Sodium Dodecyl Sulfate–Polyacrylamide Gels at the Low-Picomole Level. Application to N-Terminal Sequencing of a Scarce Protein and to In-Solution Biological Activity Analysis of On-Gel Renatured Proteins

A simple, reliable procedure for practically quantitative (90–98%) and fast (<30 min) elution of proteins from SDS-PA gels is described with reproducible recoveries in the range from 100 to 1 pmol per band, which does not require the inclusion of detergents in the elution buffer. It consists in the combination of (1) highly sensitive on-gel protein detection (50 mol per band) with imidazole-SDS-zinc (reverse staining), (2) crushing of the protein band to produce 32-m gel particles, and (3) vortexing of the slurry in a solution of a zinc-complexing agent, e.g. glycine 0.5 M or EDTA 100 mM (100 l for a 100-pmol BSA band), at room temperature. Eluted proteins can be directly analyzed by RP-HPLC, quantitatively loaded onto a PVDF membrane, or, provided that they are previously renatured on-gel, analyzed by biological activity tests. The application of the procedure to in-solution enrichment of scarce proteins for N-terminal analysis is shown.     

Membrane protein reconstitution for functional and structural studies

Membrane proteins are involved in various critical biological processes,and studying membrane proteins represents a major challenge in protein biochemistry.As shown by both structural and functional studies,the membrane environment plays an essential role for membrane proteins.In vitro studies are reliant on the successful reconstitution of membrane proteins.This review describes the interaction between detergents and lipids that aids the understanding of the reconstitution processes.Then the techniques of detergent removal and a few useful techniques to refine the formed proteoliposomes are reviewed.Finally the applications of reconstitution techniques to study membrane proteins involved in Ca2+ signaling are summarized.     

The subunit structure of bovine heart mitochondrial transhydrogenase

Reaction of purified bovine heart transhydrogenase with bifunctional cross-linking reagents dimethyl adipimidate, dimethyl pimelimidate, dimethyl suberimidate, and dithiobis(succinimidyl propionate) results in the appearance of a dimer band on sodium dodecyl sulfate polyacrylamide gels with no higher oligomers formed. Treatment of the enzyme with 6 M urea led to inactivation and prevented cross-linking by dimethyl suberimidate. Transhydrogenase reconstituted into phosphatidylcholine proteoliposomes also yielded a dimer band on cross-linking. These data indicate that soluble and functionally reconstituted transhydrogenase possesses a dimeric structure.