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Electrophoretic mobility of a monotopic membrane protein inserted into the top of supported lipid bilayers.

We have studied the translational migration of a monotopic membrane protein, the bacterial sulfide quinone reductase (SQR) in supported n-bilayers ([Formula: see text]) under the influence of an electric field parallel to the membrane plane. The direction of the migration changes when the charge of the protein changes its sign. Measuring mobilities at different pH enables us to gain experimental physico-chemical data on SQR as its isoelectric point and its estimated oligomeric state (at least trimeric) when inserted in a lipid membrane. Consequently, in addition to the migration study of membrane proteins in a lipid environment, this experimental system, previously used with a transmembrane protein, is thus suitable to define membrane protein properties in conditions approaching the native ones (in the absence of detergent).

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive.

Universal Stress Protein Regulates Electron Transfer and Superoxide Generation Activities of the Cytochrome bc1 Complex from Rhodobacter sphaeroides.

Interactions between Rhodobacter sphaeroides cytochrome bc1 complex (Rsbc1) and soluble cytosolic proteins were studied by a precipitation pull-down technique. After being purified, detergent-dispersed Rsbc1 complex was incubated with soluble cytosolic fraction and then dialyzed in the absence of detergent; the interacting proteins were coprecipitated with Rsbc1 complex upon centrifugation. One of the cytosolic proteins pulled down by Rsbc1 complex was identified by liquid chromatography-coupled tandem mass spectrometry (LC/MS/MS) to be the reported R. sphaeroides universal stress protein (UspA). Incubating purified UspA with the detergent dispersed bc1 complex resulted in an increase in the Rsbc1 complex activity by 60% and a decrease in superoxide generation activity by the complex by more than 70%. These UspA effects were only observed with Rsbc1 complexes containing subunit IV and assayed under aerobic conditions. These results suggest that the interaction between UspA and Rsbc1 complex may play an important role in R. sphaeroides cells during oxidative stress. Using a biotin label transfer technique, cytochrome c1 of the Rsbc1 complex was identified as the interacting site for UspA.

Chaperone activity of immunoglobulin-binding protein from Yersinia pseudotuberculosis

The chaperone activity of the Yersinia pseudotuberculosis immunoglobulin-binding protein Skp has been studied. Its ability to bind the unfolded proteins of the bacterial outer membrane (OMP) was found by means of Western blotting using peroxidase-labeled Skp. Using dynamic light scattering, we have shown that Skp exerts an inhibitory effect on the aggregation of unfolded recombinant OmpF, which occurs after a 10-fold dilution of the porin solution. The results obtained demonstrate that Skp prevents OMP aggregation in aqueous solutions in the absence of detergents.

Assembly and characterization of gp160-nanodiscs: A new platform for biochemical characterization of HIV envelope spikes

The human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS) and is thus responsible for significant morbidity and mortality worldwide. Despite considerable effort, preparation of an effective vaccine for AIDS has been elusive and it has become clear that a fundamental understanding of the relevant antigenic targets on HIV is essential. The Env trimer spike is the only viral antigen present on the surface of the viral particle and it is the target of all broadly neutralizing antibodies isolated to date. Thus, a soluble, homogeneous, and well-defined preparation of Env trimers is an important first step toward biochemical and structural characterization of the antigenic spike. Phospholipid bilayer nanodiscs represent a relatively new technology that can serve as a platform for the assembly of membrane proteins into a native membrane-like environment. Here we describe the preparation and characterization of unprocessed full-length, natively glycoslyated gp160 Env proteins incorporated into nanodiscs (gp160-ND). The particles are soluble and well defined in the absence of detergent, and possess a morphology anticipated of Env incorporated into a lipid ND. Importantly, the gp160-NDs retain CD4 and Env antibody binding characteristics expected of a functional trimer spike and their incorporation into a lipid membrane allows interrogation of epitopes associated with the membrane-proximal ectodomain region of gp41. These studies provide the groundwork for the use of gp160-ND in more detailed biochemical and structural studies that may set the stage for their use in vaccine development.

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.

Principal FindingsHEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025–0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the “wobble in cone” model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye.SummaryLimited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.

Novel Application of Cucurbita Maxima Peel Protease in Detergents

The protease enzyme from Cucurbita maxima peel was isolated and purified and its application in removing blood stain was studied. The optimum pH and temperature for this newly isolated protease was found to be 7.0 and 40°C respectively. The molecular weight of isolated protease was determined by SDS PAGE and it was found to be 31 KD. This new protease enzyme was applied to blood stained cloth to evaluate the stain removing capacity of the enzyme in the presence and absence of detergents. From the results, it is confirmed that the newly isolated protease removes stain completely with detergents and even in the absence of detergents. This study confirms the application of Cucurbita maxima peel protease in removing blood stains

Isolation and characterization of recombinant murine Wnt3a

Wnt proteins are a family of morphogens that possess potent biological activity. Structure–function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu2+-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt–cyclodextrin complexes may facilitate structure–activity studies in the absence of adverse detergent effects.

The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy

Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving >3.5Å resolution detail in membrane proteins of modest (~300kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function.

Isolation of Escherichia coli mannitol permease, EIImtl, trapped in amphipol A8-35 and fluorescein-labeled A8-35.

Amphipols (APols) are short amphipathic polymers that keep integral membrane proteins water-soluble while stabilizing them as compared to detergent solutions. In the present work, we have carried out functional and structural studies of a membrane transporter that had not been characterized in APol-trapped form yet, namely EII(mtl), a dimeric mannitol permease from the inner membrane of Escherichia coli. A tryptophan-less and dozens of single-tryptophan (Trp) mutants of this transporter are available, making it possible to study the environment of specific locations in the protein. With few exceptions, the single-Trp mutants show a high mannitol-phosphorylation activity when in membranes, but, as variance with wild-type EII(mtl), some of them lose most of their activity upon solubilization by neutral (PEG- or maltoside-based) detergents. Here, we present a protocol to isolate these detergent-sensitive mutants in active form using APol A8-35. Trapping with A8-35 keeps EII(mtl) soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EII(mtl) mutant was found to be ~3× higher than the activity of the same protein in dodecylmaltoside. The preparations are suitable both for functional and for fluorescence spectroscopy studies. A fluorescein-labeled version of A8-35 has been synthesized and characterized. Exploratory studies were conducted to examine the environment of specific Trp locations in the transmembrane domain of EII(mtl) using Trp fluorescence quenching by water-soluble quenchers and by the fluorescein-labeled APol. This approach has the potential to provide information on the transmembrane topology of MPs.

Synthesis, Characterization and Applications of a Perdeuterated Amphipol

Amphipols are short amphipathic polymers that can substitute for detergents at the hydrophobic surface of membrane proteins (MPs), keeping them soluble in the absence of detergents while stabilizing them. The most widely used amphipol, known as A8-35, is comprised of a polyacrylic acid (PAA) main chain grafted with octylamine and isopropylamine. Among its many applications, A8-35 has proven particularly useful for solution-state NMR studies of MPs, for which it can be desirable to eliminate signals originating from the protons of the surfactant. In the present work, we describe the synthesis and properties of perdeuterated A8-35 (perDAPol). Perdeuterated PAA was obtained by radical polymerization of deuterated acrylic acid. It was subsequently grafted with deuterated amines, yielding perDAPol. The number-average molar mass of hydrogenated and perDAPol, ~4 and ~5 kDa, respectively, was deduced from that of their PAA precursors, determined by size exclusion chromatography in tetrahydrofuran following permethylation. Electrospray ionization-ion mobility spectrometry-mass spectrometry measurements show the molar mass and distribution of the two APols to be very similar. Upon neutron scattering, the contrast match point of perDAPol is found to be ~120% D2O. In (1)H-(1)H nuclear overhauser effect NMR spectra, its contribution is reduced to ~6% of that of hydrogenated A8-35, making it suitable for extended uses in NMR spectroscopy. PerDAPol ought to also be of use for inelastic neutron scattering studies of the dynamics of APol-trapped MPs, as well as small-angle neutron scattering and analytical ultracentrifugation.

In vivo characterization of the biodistribution profile of amphipol A8-35.

Amphipols (APols) are polymeric surfactants that keep membrane proteins (MPs) water-soluble in the absence of detergent, while stabilizing them. They can be used to deliver MPs and other hydrophobic molecules in vivo for therapeutic purposes, e.g., vaccination or targeted delivery of drugs. The biodistribution and elimination of the best characterized APol, a polyacrylate derivative called A8-35, have been examined in mice, using two fluorescent APols, grafted with either Alexa Fluor 647 or rhodamine. Three of the most common injection routes have been used, intravenous (IV), intraperitoneal (IP), and subcutaneous (SC). The biodistribution has been studied by in vivo fluorescence imaging and by determining the concentration of fluorophore in the main organs. Free rhodamine was used as a control. Upon IV injection, A8-35 distributes rapidly throughout the organism and is found in most organs but the brain and spleen, before being slowly eliminated (10-20 days). A similar pattern is observed after IP injection, following a brief latency period during which the polymer remains confined to the peritoneal cavity. Upon SC injection, A8-35 remains essentially confined to the point of injection, from which it is only slowly released. An interesting observation is that A8-35 tends to accumulate in fat pads, suggesting that it could be used to deliver anti-obesity drugs.

Stabilizing hyperactivated lecitase structures through physical treatment with ionic polymers

Lecitase Ultra has been covalently immobilized on cyanogen bromide cross-linked 4% agarose (CNBr) beads, maintaining 70% of the initial activity. The activity of the immobilized enzyme was improved in the presence of Triton X-100, sodium dodecyl sulfate (SDS), and cetyltrimethyl ammonium bromide (CTAB) (e.g., up to 800% when using CTAB). However, CTAB and Triton X-100 presented a negative effect on enzyme stability even at low concentrations, and SDS cannot be used for a long time at 1% concentration. To maintain the hyperactivated conformation of the enzyme in the absence of detergent, ionic polymers were added during incubation of the immobilized enzyme in the presence of detergents. Coating the immobilized enzyme with polyethylenimine in aqueous buffer (PEI) produced a 3-fold increase in enzyme activity. However, in the presence of 0.1% SDS (v/v), this coating produced a 50-fold increase in enzyme activity. Using PEI and 0.01% (v/v) CTAB, the Lecitase activity decreased to 10%. Using irreversible inhibitors, it could be shown that the PEI/SDS-CNBr-Lecitase preparation allowed its catalytic Ser to be more accessible to the reaction medium than the unmodified CNBr-Lecitase.

Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins.

Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8–35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting ‘OligAPol’ have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8–35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications.

Contributing ratio of the mechanical and the physicochemical actions to washing efficiency of mineral oil and protein soils

It was attempted to separate the detergency contributions from the mechanical actions and the physicochemical actions for two different types of soils. The mechanical washing efficiency was evaluated through the washing performed under the mechanical agitation, in the absence of detergent. The physicochemical washing efficiency was examined in the washing bath with detergent in the absence of mechanical agitation. Among the washing parameters, extended washing time and higher washing temperature were effective improving the mechanical and physicochemical washing efficiencies for mineral oil and carbon black. For blood soil, lower washing temperature and extended washing time enhanced the mechanical and physicochemical washing efficiencies. For the mineral oil, the presence of both mechanical and physicochemical actions led to the effective washing performance with synergistic effect. For the blood soil, mechanical actions were observed as more contributing than the physicochemical actions. The optimized washing program could be recommended as per the contributing ratios for the specific soil types, minimizing the energy consumption and detergent usage while achieving the washing performance.

Detergent-free mass spectrometry of membrane protein complexes.

The rapid growth in the study of membrane protein complexes in lipid microenvironments prompts new methods for their characterization. Here we develop mass spectrometry as a method for studying membrane proteins incorporated into amphipols, bicelles and nanodiscs. We compare these detergent-free vehicles with micelles for their ability to preserve the interactions of oligomeric complexes and to stabilize proteins that require defined lipid environments.

Influences on Trimerization and Aggregation of Soluble, Cleaved HIV-1 SOSIP Envelope Glycoprotein

We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.

Methodological study on the removal of solid oil and fat stains from cotton fabrics using abrasion

Solid fats are one of the most difficult stains to remove at low temperatures. Mechanical action is beneficial for stain removal, but the potential and limitations of such an essential part of washing are not known. Fabric abrasion has been studied for the first time in a systematic and controlled manner using a tribometer. The efficiency of cotton–cotton abrasion, to simulate the rubbing of clothes, was studied in the absence of detergents using models of liquid and solid oils (hexadecane, octadecane, and undecanoic acid) and real fats (lard and buttermilk fat). In model oils, abrasion is not very effective at any temperature, whereas in typical fats abrasion significantly improves cleaning in a wide range of temperatures. The different behavior is caused by the temperature-dependent solid fat content of lard and butter. Fluorescence microscopy is introduced as a novel methodology for the quantification of the fat content in soiled fabrics.

Characterization of human β,β-carotene-15,15′-monooxygenase (BCMO1) as a soluble monomeric enzyme

The formal first step in in vitamin A metabolism is the conversion of its natural precursor β,β-carotene (C40) to retinaldehyde (C20). This reaction is catalyzed by the enzyme β,β-carotene-15,15′-monooxygenase (BCMO1). BCMO1 has been cloned from several vertebrate species, including humans. However, knowledge about this protein’s enzymatic and structural properties is scant. Here we expressed human BCMO1 in Spodoptera frugiperda 9 insect cells. Recombinant BCMO1 is a soluble protein that displayed Michaelis–Menten kinetics with a KM of 14μM for β,β-carotene. Though addition of detergents failed to increase BCMO1 enzymatic activity, short chain aliphatic detergents such as C8E4 and C8E6 decreased enzymatic activity probably by interacting with the substrate binding site. Thus we purified BCMO1 in the absence of detergent. Purified BCMO1 was a monomeric enzymatically active soluble protein that did not require cofactors and displayed a turnover rate of about 8 molecules of β,β-carotene per second. The aqueous solubility of BCMO1 was confirmed in mouse liver and mammalian cells. Establishment of a protocol that yields highly active homogenous BCMO1 is an important step towards clarifying the lipophilic substrate interaction, reaction mechanism and structure of this vitamin A forming enzyme.

Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35

Amphipols are a class of amphipathic polymers designed to maintain membrane proteins in aqueous solutions in the absence of detergents. Denatured β-barrel membrane proteins, like outer membrane proteins OmpA from Escherichia coli and FomA from Fusobacterium nucleatum, can be folded by dilution of the denaturant urea in the presence of amphipol A8-35. Here, the folding kinetics and stability of OmpA in A8-35 have been investigated. Folding is well described by two parallel first-order processes, whose half-times, ~5 and ~70 min, respectively, are independent of A8-35 concentration. The faster process contributed ~55-64 % to OmpA folding. Folding into A8-35 was faster than into dioleoylphosphatidylcholine bilayers and complete at ratios as low as ~0.17 g/g A8-35/OmpA, corresponding to ~1-2 A8-35 molecules per OmpA. Activation energies were determined from the temperature dependence of folding kinetics, monitored both by electrophoresis, which reports on the formation of stable OmpA tertiary structure, and by fluorescence spectroscopy, which reflects changes in the environment of tryptophan side chains. The two methods yielded consistent estimates, namely ~5-9 kJ/mol for the fast process and ~29-37 kJ/mol for the slow one, which is lower than is observed for OmpA folding into dioleoylphosphatidylcholine bilayers. Folding and unfolding titrations with urea demonstrated that OmpA folding into A8-35 is reversible and that amphipol-refolded OmpA is thermodynamically stable at room temperature. Comparison of activation energies for folding and unfolding in A8-35 versus detergent indicates that stabilization of A8-35-trapped OmpA against denaturation by urea is a kinetic, not a thermodynamic phenomenon.