Detergent Removal Research Articles

Current trends in the production and biomedical applications of liposomes: a review

A review of literature was carried out to determine methods of production of liposomes, their stability, biodistribution and their uses as drug delivery systems. The conventional method of preparing liposomes is basically for the multilamellar vesicles (MLVs). However, other methods are used to reduce the size of these MLVs to small unilamellar vesicles (SUVs) so as to increase their plasma lifetime and consequently increase the possibility of achieving greater tissue localisation. Some of these methods of size reduction are sonication and high pressure extrusion. Each of these methods has its own advantages and disadvantages. Large unilamellar vesicles (LUVs), on the other hand, are prepared mainly by detergent removal method and reverse phase extrusion technique. There are also improved pharmacokinetic properties with liposomal drugs compared to free drugs, though some formulation factors affect the release kinetics of the liposomal drugs. The review also shows that liposomes have a lot of biomedical applications and uses. They have been used in drug targeting, oral delivery of vaccines, insulins, peptides and some compounds, which are usually degraded in the gastrointestinal tract. It has also found application in topical therapy especially in the eye and lungs. Other areas of application are in cancer chemotherapy and treatment of human immunovirus (HIV) infection. The control of the stability of liposomes is an essential pre-requisite for effective use as drug carriers. Leakage of the liposome is attributable mainly to differences in lamellar structure. For instance, MLVs are less prone to leakage than ULVs. The use of a combination of saturated phospholipid and cholesterol in the formulation of the liposomes has also been found to enhance stability with lower tendency to leakage. Keywords: Liposomes,phospholipid, encapsulation, applications Journal of Medicine and Biomedical Research Vol. 4 (1) 2005: pp. 9-21

Solid-State NMR Study of the Human Peripheral Cannabinoid Receptor CB2 in Lipid Bilayers

Human peripheral cannabinoid receptor CB2 expressed in E. coli. has been purified, and successfully reconstituted in functional form into lipid bilayers composed of phosphatidylcholine, phosphatidylserine, and cholesteryl hemisuccinate (CHS). The reconstitution was carried out by detergent removal from the CB2-lipid-detergent mixed micelles on an adsorbent column, or by rapid dilution below the critical micelle concentration followed by washing on a concentrator. Proteoliposomes prepared at the CB2:phospholipid molar ratio of 1:600 showed the following basic physical properties: Free of detergents (as analyzed by high-resolution 1H NMR), homogeneity of the CB2-to-lipid ratio over the proteoliposome particles (by sucrose gradient centrifugation), unilamellarity with a mean diameter of ∼150-200 nm (by dynamic light scattering), and functional integrity (by G-protein activation assay). Using the proteoliposomes, CB2-lipid interactions were investigated by solid-state NMR. Order parameters of the phospholipid acyl chains measured by 2H quadrupolar splittings indicated that CHS, a detergent-soluble analog of cholesterol, preferentially interacts with the 1-palmitoyl-2-oleoyl-sn-glycero-3-phospholipids over CB2. By probing 1H NMR saturation transfer, evidence for CB2-lipid interactions at the lipid acyl chains and less significant interactions at the glycerol backbone and the headgroups were observed. 1H spin-lattice relaxation rates decreased notably at the acyl-chains upon CB2 incorporation, indicating reduced motion on the nanosecond timescale corresponding to the restriction of phospholipid wobbling about the bilayer normal. Structure-function relationships in view of the role of interactions between CB2 and anionic phosphatidylserine in activation of G-protein will also be discussed.

Role of the C Terminus of the Photoreceptor ABCA4 Transporter in Protein Folding, Function, and Retinal Degenerative Diseases

ABCA4 is an ATP-binding cassette transporter that is expressed in rod and cone photoreceptor cells and implicated in the removal of retinal derivatives from outer segments following photoexcitation. Mutations in the ABCA4 gene are responsible for a number of related retinal degenerative diseases, including Stargardt macular degeneration, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. In order to determine the role of the C terminus of ABCA4 in protein structure and function and understand mechanisms by which C-terminal mutations cause retinal degenerative diseases, we have expressed and purified a series of deletion and substitution mutants of ABCA4 and ABCA1 in HEK 293T cells for analysis of their cellular localization and biochemical properties. Removal of the C-terminal 30 amino acids of ABCA4, including a conserved VFVNFA motif, resulted in a loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal-stimulated ATPase activity. This mutant was also retained in the endoplasmic reticulum of cells. Replacement of the VFVNFA motif with alanine residues also resulted in loss in function and cellular mislocalization. In contrast, C-terminal deletion mutants that retain the VFVNFA motif were functionally active and localized to intracellular vesicles similar to wild-type ABCA4. Our studies indicated that the VFVNFA motif is required for the proper folding of ABCA4 into a functionally active protein. This motif also contributes to the efficient folding of ABCA1 into an active protein. Our results provide a molecular based rationale for the disease phenotype displayed by individuals with mutations in the C terminus of ABCA4.

Citations

Citations

Characterization of membrane protein reconstitution in LUVs of different lipid composition by fluorescence anisotropy

A major requirement to perform structural studies with membrane proteins is not only to define efficient reconstitution protocols, that assure a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. For this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. In this study we report the reconstitution of OmpF in preformed DMPC and E. coli liposomes using two different techniques for detergent removal: (1) exclusion chromatography and (2) incubation with detergent-adsorbing beads. The incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine OmpF effect on the structural order of membrane lipids. These results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. Furthermore, it is possible to state that although the insertion is directly related to the size distributions of proteoliposomes, it could be efficiently recognized by steady-state fluorescence anisotropy. This technique, more popular among cell biologists, can be a very practical and straightforward alternative to DLS to confirm membrane protein insertion.

Induction of Efficient Energy Dissipation in the Isolated Light-harvesting Complex of Photosystem II in the Absence of Protein Aggregation

Under excess illumination, the Photosystem II light-harvesting antenna of higher plants has the ability to switch into an efficient photoprotective mode, allowing safe dissipation of excitation energy into heat. In this study, we show induction of the energy dissipation state, monitored by chlorophyll fluorescence quenching, in the isolated major light-harvesting complex (LHCII) incorporated into a solid gel system. Removal of detergent caused strong fluorescence quenching, which was totally reversible. Singlet-singlet annihilation and gel electrophoresis experiments suggested that the quenched complexes were in the trimeric not aggregated state. Both the formation and recovery of this quenching state were inhibited by a cross-linker, implying involvement of conformational changes. Absorption and CD measurements performed on the samples in the quenched state revealed specific alterations in the spectral bands assigned to the red forms of chlorophyll a, neoxanthin, and lutein 1 molecules. The majority of these alterations were similar to those observed during LHCII aggregation. This suggests that not the aggregation process as such but rather an intrinsic conformational transition in the complex is responsible for establishment of quenching. 77 K fluorescence measurements showed red-shifted chlorophyll a fluorescence in the 690-705 nm region, previously observed in aggregated LHCII. The fact that all spectral changes associated with the dissipative mode observed in the gel were different from those of the partially denatured complex strongly argues against the involvement of protein denaturation in the observed quenching. The implications of these findings for proposed mechanisms of energy dissipation in the Photosystem II antenna are discussed.

Liposomes incorporating hydrophobically modified glucose oxidase

Glucose oxidase (GOD) was hydrophobically modified by conjugating palmitic acids to the amino groups of the enzyme. The number of palmitic acid residues conjugated to GOD was determined by using trinitrobenzenesulfonic acid (TNBS). According to the result, six palmitic acids turned out to be covalently attached to one GOD molecule. The activity of the modified GOD was 75%–80% of that of the native one, and due to the conjugated alkyl chain, the modified GOD was more surface-active than the native one. The modified GOD was incorporated into the liposomal bilayer of dioleoylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) by a detergent removal method. According to the results of the pH-dependent release, no significant release was observed in 60 sec at pH 8.2. An appreciable amount of calcein released at pH 7.0. A marked amount released at pH 6.0. The degree of release became more extensive at pH 5.0 and pH 5.6. This is possibly because the liposome is disintegrated into hexagonal phases under acidic conditions.

Overexpression and Functional Characterization of the Extracellular Domain of the Human α1 Glycine Receptor

A novel truncated form (residues 1-214, with a randomized C-terminal tail) of the ligand-binding extracellular domain (ECD) of the human alpha1 glycine receptor (GlyR), with amino acids from the corresponding sequence of an acetylcholine binding protein (AChBP) substituted for two relatively hydrophobic membrane-proximal loops, was overexpressed using a baculovirus expression system. The mutant GlyR ECD, named GlyBP, was present in both soluble and membrane-associated fractions after cell lysis, though only the latter appeared to be in a native-like conformation capable of binding strychnine, a GlyR specific antagonist. The membrane-associated GlyBP was solubilized, and detergent/lipid/protein micelles were affinity purified. After detergent removal, GlyBP may be isolated in either aqueous or vesicular form. Binding assays and spectroscopic studies using circular dichroism and FRET are consistent with both forms adopting equivalent native-like conformations. Thus, GlyBP may be isolated as a soluble or membrane-associated assembly that serves as a structural and functional homologue of the ECD of GlyR.

Removal of detergents from protein digests for mass spectrometry analysis

Detergents are commonly used for the extraction of hydrophobic proteins and must be removed for sensitive detection of peptides by mass spectrometry. We demonstrate that ethyl acetate is able to extract octylglycoside from a protease digest without loss of peptides or interference with the peptide mass spectral profile. Ethyl acetate extraction was also found to reduce interference by sodium dodecyl sulfate, Nonidet P-40, or Triton X-100 in the mass spectrometry analysis.

Direct and Potent Regulation of γ-Secretase by Its Lipid Microenvironment

gamma-Secretase is an unusual and ubiquitous aspartyl protease with an intramembrane catalytic site that cleaves many type-I integral membrane proteins, most notably APP and Notch. Several reports suggest that cleavage of APP to produce the Abeta peptide is regulated in part by lipids. As gamma-secretase is a multipass protein complex with 19 transmembrane domains, it is likely that the local lipid composition of the membrane can regulate gamma-activity. To determine the direct contribution of the lipid microenvironment to gamma-secretase activity, we purified the human protease from overexpressing mammalian cells, reconstituted it in vesicles of varying lipid composition, and examined the effects of individual phospholipids, sphingolipids, cholesterol, and complex lipid mixtures on substrate cleavage. A conventional gamma-activity assay was modified to include a detergent-removal step to facilitate proteoliposome formation, and this increased baseline activity over 2-fold. Proteoliposomes containing sphingolipids significantly increased gamma-secretase activity over a phosphatidylcholine-only baseline, whereas the addition of phosphatidylinositol significantly decreased activity. Addition of soluble cholesterol in the presence of phospholipids and sphingolipids robustly increased the cleavage of APP- and Notch-like substrates in a dose-dependent manner. Reconstitution of gamma-secretase in complex lipid mixtures revealed that a lipid raft-like composition supported the highest level of activity compared with other membrane compositions. Taken together, these results demonstrate that membrane lipid composition is a direct and potent modulator of gamma-secretase and that cholesterol, in particular, plays a major regulatory role.

The use of detergents to purify membrane proteins.

Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, or biomolecular NMR. Protocols in this unit include the isolation and solubilization of biological membranes, phase separation, and support protocols for detergent removal and detergent exchange using different methods.

Glycosaminoglycan-resistant and pH-sensitive lipid-coated DNA complexes produced by detergent removal method

Cationic polymers are efficient gene delivery vectors in in vitro conditions, but these carriers can fail in vivo due to interactions with extracellular polyanions, i.e. glycosaminoglycans (GAG). The aim of this study was to develop a stable gene delivery vector that is activated at the acidic endosomal pH. Cationic DNA/PEI complexes were coated by 1,2-dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) (3:2 mol/mol) using two coating methods: detergent removal and mixing with liposomes prepared by ethanol injection. Only detergent removal produced lipid-coated DNA complexes that were stable against GAGs, but were membrane active at low pH towards endosome mimicking liposomes. In relation to the low cellular uptake of the coated complexes, their transfection efficacy was relatively high. PEGylation of the coated complexes increased their cellular uptake but reduced the pH-sensitivity. Detergent removal was thus a superior method for the production of stable, but acid activatable, lipid-coated DNA complexes.

Detergent Removal from Protein Samples Using SDR HyperD ® and Mass-Spectrometry Based Detergent Estimation

Detergents are widely used in protein chemistry protocols and may be necessary for protein extraction, solubilization and denaturation. However, the presence of detergents is known to interfere with many techniques including Massspectrometry (MS), hence their removal is a pre-requisite for error-free MS analyses. This study evaluates the use of SDR HyperD ® , a unique chromatography resin, for the removal of NP-40, Triton X-100 and CHAPS detergents from protein samples. Additionally, a highly sensitive (detection limit 100 ppm) MS based method for detection and quantitation of these detergents is described. SDR HyperD ® is used in combination with Nanosep ® spin devices format, readily amenable for high-throughput applications. SDR HyperD ® treatment results in substantial improvement in MS signal-intensity of proteins. Estimation of residual detergents in treated samples establishes that SDR

Reconstitution into liposomes of the B°-like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na + dependent [ 3H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K +. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [ 3H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na +and internal K +. The transporter showed low if there is any tolerance towards the substitution of Na + or K + for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na + concentration cooperativity index close to 1 was derived, indicating that 1 Na + should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na + transport. Optimal rate of 0.1 mM [ 3H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5–7.0. [ 3H]glutamine (and [ 3H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl 2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue α-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na + dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B°-like.

Mechanism of Micelle-Vesicle Transformation and Control of Vesicular Sizes and Properties

The mechanism of vesicle-to-micelle or micelle-to-vesicle transition was studied in order to control sizes and fluidities of vesicles during periods of preparation. Dependence of particle sizes measured by quasi-elastic light scattering, turbidities, fluidity parameters monitored by ESR spectroscopy, and morphological changes of mixed aggregates of egg yolk phosphatidylcholine (EPC) and a detergent (octylglucoside (OG) or sodium cholate (Na-chol)) on detergent concentration provided a model of vesicle destruction. It possessed three phase transition points, and proceeded in a stepwise fashion: vesicles, small particles containing large amounts of detergents (SUV(*)), intermediate structures, and mixed micelles. Vesicle formation on removal of detergents from micelles proceeded oppositely. Micelle-vesicle transition mechanism was common to all detergents examined. The feature of the mechanism was the presence of SUV(*). Next, SUV(*) was prepared by adding appropriate amount of a detergent to small unilamellar vesicles obtained by sonication. Time-dependent size growth of the SUV(*) was remarkable in the case of OG-containing SUV(*), but was insignificant in the case of Na-chol-containing SUV(*), suggesting the size determining step to be the stage of the SUV(*). The tendency to produce large or small vesicles from micelles was related to the absence or presence, respectively, of a net charge in the detergent molecule. The fluidities of EPC micelles containing small amounts of a detergent possessing a steroidal structure (e.g., Na-chol or CHAPS) were significantly smaller than the corresponding values of a detergent without a steroidal structure (e.g., OG), suggesting a method of control of orderliness of hydrocarbon chains in EPC vesicles.

Colicin N Binds to the Periphery of Its Receptor and Translocator, Outer Membrane Protein F

SummaryColicins kill Escherichia coli after translocation across the outer membrane. Colicin N displays an unusually simple translocation pathway, using the outer membrane protein F (OmpF) as both receptor and translocator. Studies of this binary complex may therefore reveal a significant component of the translocation pathway. Here we show that, in 2D crystals, colicin is found outside the porin trimer, suggesting that translocation may occur at the protein-lipid interface. The major lipid of the outer leaflet interface is lipopolysaccharide (LPS). It is further shown that colicin N binding displaces OmpF-bound LPS. The N-terminal helix of the pore-forming domain, which is not required for pore formation, rearranges and binds to OmpF. Colicin N also binds artificial OmpF dimers, indicating that trimeric symmetry plays no part in the interaction. The data indicate that colicin is closely associated with the OmpF-lipid interface, providing evidence that this peripheral pathway may play a role in colicin transmembrane transport.

PH-dependent release property of dioleoylphosphatidyl ethanolamine liposomes

pH-sensitive liposomes were prepared by a detergent removal method. Dioleoylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) were combined for the preparation of the liposomes so that the molar ratios of DOPE to CHEMS are 9/1, 8/2, 6/4 and 5/5. On transmission electron micrographs, hundreds of nm sized-multilamella vesicles were observed. The degrees of fluorescence quenching were approximately 70–80%, indicating that closed vesicles were formed. According to the results of the pH-dependent release experiment with the liposome composed of DOPE/CHEMS (5/5), no significant release was observed in the pH region ranging from 6 to 8. At pH of 5, an appreciable amount of calcein was released. The patterns of pH-dependent releases from liposomes composed of DOPE/CHEMS (6/4) and DOPE/CHEMS (8/2) were almost the same as those from liposomes composed of DOPE/ CHEMS (5/5). With the liposomes composed of DOPE/CHEMS (9/1), unlike the other liposomes described above, almost 90% release was observed at pH 6. In this case, since the amount of a complementary molecule, CHEMS, is relatively small, the liposomes would be subjective to destabilization even at a small change in the degree of deionization of the carboxylic group. This may explain why the liposome of DOPE/CHEMS (9/1) exhibits a significant release at a relatively high pH, pH 6.0.

The Key Residue for Substrate Transport (Glu14) in the EmrE Dimer Is Asymmetric

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.

Single-step immunoaffinity purification and functional reconstitution of human phagocyte flavocytochrome b

Human neutrophil flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that generates high levels of superoxide in the multisubunit NADPH oxidase complex. Since Cyt b is currently isolated in limited quantities, improved methods for purification from low levels of starting membranes (from both neutrophils and other expressing cell types) are important for the analysis of structure and catalytic mechanism. In the present study, the epitope-mapped monoclonal antibody CS9 was coupled to Sepharose beads and used as an affinity matrix for single-step immunoaffinity purification of Cyt b. Following solubilization of both human neutrophil and PLB-985 membrane fractions in the nonionic detergent octylglucoside, Cyt b was absorbed on the CS9-Sepharose affinity matrix and purified protein was eluted under non-denaturing conditions with an epitope-mimicking peptide. The high efficiency of this isolation procedure allowed Cyt b to be reproducibly purified from readily obtainable levels of starting membrane fractions (9×108 cell equivalents of neutrophil membranes and 2×109 cell equivalents of PLB-985 membranes). Since Cyt b could be affinity-purified in the detergent octylglucoside, high-level functional reconstitution was carried out directly on elution fractions by simple addition of solubilized phospholipid and subsequent dialysis for detergent removal. To our knowledge, this study describes the most efficient method for generating purified, functionally-reconstituted Cyt b and should facilitate analyses that require a highly-defined NADPH oxidase system.

Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis

Mitochondrial F 1F o-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F 1F o-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F 1F o-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F 1F o-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F 1 catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F 1 to the lipid monolayer and the F o membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F o by electron microscopy and AFM.