Effects Of Different Detergents Research Articles

Optimization of Membrane Protein TmrA Purification Procedure Guided by Analytical Ultracentrifugation.

Membrane proteins are involved in various cellular processes. However, purification of membrane proteins has long been a challenging task, as membrane protein stability in detergent is the bottleneck for purification and subsequent analyses. Therefore, the optimization of detergent conditions is critical for the preparation of membrane proteins. Here, we utilize analytical ultracentrifugation (AUC) to examine the effects of different detergents (OG, Triton X-100, DDM), detergent concentrations, and detergent supplementation on the behavior of membrane protein TmrA. Our results suggest that DDM is more suitable for the purification of TmrA compared with OG and TritonX-100; a high concentration of DDM yields a more homogeneous protein aggregation state; supplementing TmrA purified with a low DDM concentration with DDM maintains the protein homogeneity and aggregation state, and may serve as a practical and cost-effective strategy for membrane protein purification.

Influence of Detergent and Lipid Composition on Reconstituted Membrane Proteins for Structural Studies.

Membrane proteins are frequently reconstituted in different detergents as a prerequisite to create a phospholipid environment reminiscent of their native environment. Different detergent characteristics such as their chain length and bond types could affect the structure and function of proteins. Yet, they are seldom taken into account when choosing a detergent for structural studies. Here, we explore the effect of different detergents and lipids with varying degrees of double- or single-bond composition on 1H–15N transverse relaxation optimized spectroscopy spectra of the outer membrane protein W (OmpW). We observed changes in nuclear magnetic resonance chemical shifts for OmpW reconstituted in micelles, bicelles, and nanodiscs, depending on their detergent/lipid composition. These results suggest that a careful evaluation of detergents is necessary, so as not to jeopardize the structure and function of the protein.

The Det.Belt Server: A Tool to Visualize and Estimate Amphipathic Solvent Belts around Membrane Proteins.

Detergents wrap around membrane proteins to form a belt covering the hydrophobic part of the protein serving for membrane insertion and interaction with lipids. The number of detergent monomers forming this belt is usually unknown to investigators, unless dedicated detergent quantification is undertaken, which for many projects is difficult to setup. Yet, having an approximate knowledge of the amount of detergent forming the belt is extremely useful, to better grasp the protein of interest in interaction with its direct environment rather than picturing the membrane protein “naked”. We created the Det.Belt server to dress up membrane proteins and represent in 3D the bulk made by detergent molecules wrapping in a belt. Many detergents are included in a database, allowing investigators to screen in silico the effect of different detergents around their membrane protein. The input number of detergents is changeable with fast recomputation of the belt for interactive usage. Metrics representing the belt are readily available together with scripts to render quality 3D images for publication. The Det.Belt server is a tool for biochemists to better grasp their sample.

The Effect of Using Different Detergents in Cleaning Cows' Udders on The Microbial Content of Produced Milk

This study investigated the effect of different detergents used to clean cows' udders on the microbial content of the produced milk using twenty cows in Ajloun, a northern city in Jordan. The milking process was repeated from same cows on three successive days. On day 1, we milked the cows after cleaning their udders using water only. This was repeated on the two successive days. Thereafter, the cows were milked after cleaning their udders by a different detergent each day. The process was also repeated for three successive days for each detergent. Microbial Analysis was carried out on the collected milk samples. The results indicated that cleaning cows' udders before milking has improved the hygiene conditions and reduced the total bacterial count, total coliform, staphylococci and enterococci spp counts and the values of yeast and molds. Different detergents had different effects on the microbial counts. Finally, the effectiveness of the detergent differed according to its brand. Our findings are important to public health because milk has been a traditional food and ironically a very potent carrier of gastrointestinal infections, if contaminated.

Detergent decellularization methods affect the surface molecular functionality of biologic scaffolds

Detergent decellularization methods affect the surface molecular functionality of biologic scaffolds

Functional Reconstitution of Staphylococcus aureus Truncated AgrC Histidine Kinase in a Model Membrane System

The integral membrane protein AgrC is a histidine kinase whose sensor domains interact with an autoinducing peptide, resulting in a series of downstream responses. In this study, truncated AgrCTM5-6C and AgrCTM5-6C-GFP with GFP as a reporter gene were produced using a bacterial system. Purified AgrCTM5-6C and AgrCTM5-6C-GFP were reconstituted into liposomes by a detergent-mediated method. To achieve high-yield protein incorporation, we investigated the effect of different detergents on protein reconstitution efficiency. The highest incorporation was found with N,N-dimethyldode-cylamine N-oxide during complete liposome solubilization, which resulted in a yield of 85±5%. The COOH-terminus of the protein AgrCTM5-6C was almost exclusively oriented towards the inside of the vesicles. AgrCTM5-6C in proteoliposomes exhibited approximately a 6-fold increase in constitutive activity compared with AgrCTM5-6C in detergent micelles. The reconstitution of AgrCTM5-6C or AgrCTM5-6C-GFP was characterized using dynamic light scattering, fluorescence microscopy, and transmission electron microscopy. Based on the results, the optimal conditions for protein incorporation were defined. These findings contribute to the study of membrane protein structure and function in vitro using a reconstitution system.

Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus

Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein (GFP) fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) at yields of ≥10 mg/L, following optimization. We also assessed the effects of different detergents on membrane solubilization and AgrC kinase activity, and polyoxyethylene-(23)-lauryl-ether (Brij-35) was identified as the most suitable detergent. Furthermore, the secondary structural stability of purified AgrC was analyzed using circular dichroism (CD) spectroscopy. This study may serve as a general guide for improving the yields of other membrane protein preparations and selecting the appropriate detergent to stabilize membrane proteins for biophysical and biochemical analyses.

DIFFERENT APPROACHES TO CRYSTALLIZATION OF MEMBRANE PROTEINS

Crystallography is more like an art than science. Crystallizing membrane proteins are a big challenge; membrane proteins are present in the cell membrane and serve as cell support. The most important feature of membrane protein is that it contains both hydrophobic and hydrophilic regions on its surface. They are generally much more difficult to study than soluble proteins. The problem becomes more difficult when trying to obtain crystals to determine the high resolution structures of membrane proteins. We want to utilize this opportunity to briefly examine various approaches for crystallization of membrane proteins. The important factors for determining the success of crystallization experiments for membrane proteins lies in the purification, preparation of membrane samples, the environment in which the crystals are grown and the technique used to grow the crystals. All the X-ray structures of membrane protein are grown from preparations of detergents by different methods developed to crystallize. In this review different techniques for the crystallization of membrane proteins are being described. The cubic phase method also known as in meso method is discussed along with other methods to understand about the crystallization of membrane proteins, its general applicability, salt, detergent and screening effects on crystallization. Low volumes as nano-liter of samples can be used for crystallization. The effects of different detergents on the crystallization of membrane protein, as well as the use of surfactants like polyoxyethylene. Approach based on the detergent complexation to prove the ability of cyclodextrins to remove detergent from ternary mixtures in order to get 2D crystals. Crystallization of membrane proteins using non-ionic surfactants as well as Lipidic sponge phase and with swollen lipidic mesophases is discussed to better understand the crystallization of membrane proteins.

Crystallization chaperone strategies for membrane proteins

From G protein-coupled receptors to ion channels, membrane proteins represent over half of known drug targets. Yet, structure-based drug discovery is hampered by the dearth of available three-dimensional models for this large category of proteins. Other than efforts to improve membrane protein expression and stability, current strategies to improve the ability of membrane proteins to crystallize involve examining many orthologs and DNA constructs, testing the effects of different detergents for purification and crystallization, creating a lipidic environment during crystallization, and cocrystallizing with covalent or non-covalent soluble protein chaperones with an intrinsic high propensity to crystallize. In this review, we focus on this last category, highlighting successes of crystallization chaperones in membrane protein structure determination and recent developments in crystal chaperone engineering, including molecular display to enhance chaperone crystallizability, and end with a novel generic approach in development to target any membrane protein of interest.

Purification, immobilization, and characterization of a specific lipase from Staphylococcus warneri EX17 by enzyme fractionating via adsorption on different hydrophobic supports

Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl-Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate-polyacrylamide electrophoresis analysis gel was that corresponding to the SWL-45, which could be easily desorbed from the support by incubation with triton X-100, producing a purified enzyme. SWL-45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL-45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X-100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated.

The solubilisation of boar sperm membranes by different detergents - a microscopic, MALDI-TOF MS, 31P NMR and PAGE study on membrane lysis, extraction efficiency, lipid and protein composition

BackgroundDetergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs) from cells. Different detergents affect different membrane structures according to their physico-chemical properties. However, the effects of different detergents on membrane lysis of boar spermatozoa and the lipid composition of DRMs prepared from the affected sperm membranes have not been investigated so far.ResultsSpermatozoa were treated with the selected detergents Pluronic F-127, sodium cholate, CHAPS, Tween 20, Triton X-100 and Brij 96V. Different patterns of membrane disintegration were observed by light and electron microscopy. In accordance with microscopic data, different amounts of lipids and proteins were released from the cells by the different detergents. The biochemical methods to assay the phosphorus and cholesterol contents as well as 31P NMR to determine the phospholipids were not influenced by the presence of detergents since comparable amounts of lipids were detected in the organic extracts from whole cell suspensions after exposure to each detergent. However, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry applied to identify phospholipids was essentially disturbed by the presence of detergents which exerted particular suppression effects on signal intensities. After separation of the membrane fractions released by detergents on a sucrose gradient only Triton X-100 and sodium cholate produced sharp turbid DRM bands. Only membrane solubilisation by Triton X-100 leads to an enrichment of cholesterol, sphingomyelin, phosphatidylinositol and phosphatidylethanolamine in a visible DRM band accompanied by a selective accumulation of proteins.ConclusionThe boar sperm membranes are solubilised to a different extent by the used detergents. Particularly, the very unique DRMs isolated after Triton X-100 exposure are interesting candidates for further studies regarding the architecture of sperm.

Serial protein labeling with infrared maleimide dyes to identify cysteine modifications

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.

T‐cell antigen‐receptor stoichiometry: pre‐clustering for sensitivity

The T-cell antigen receptor (TCR x CD3) is a multi-subunit complex that is responsible for triggering an adaptive immune response. It shows high specificity and sensitivity, while having a low affinity for the ligand. Furthermore, T cells respond to antigen over a wide concentration range. The stoichiometry and architecture of TCR x CD3 in the membrane have been under intense scrutiny because they might be the key to explaining its paradoxical properties. This review highlights new evidence that TCR x CD3 is found on intact unstimulated T cells in a monovalent form (one ligand-binding site per receptor) as well as in several distinct multivalent forms. This is in contrast to the TCR x CD3 stoichiometries determined by several biochemical means; however, these data can be explained by the effects of different detergents on the integrity of the receptor. Here, we discuss a model in which the multivalent receptors are important for the detection of low concentrations of ligand and therefore confer sensitivity, whereas the co-expressed monovalent TCR x CD3s allow a wide dynamic range.

Spectroscopic study of fluorescent peptides for prenyl transferase assays

A study of the prenyl transferase reactions was performed by fluorescence using rat brain cytosol fractions as an enzyme source. Four dansylated peptides corresponding to the C-terminal sequence of Ras isoforms were synthesised. The effects of different detergents on the farnesylation or geranylgeranylation of the four peptides were evaluated. Dose-dependent effects of dodecyl-maltoside, a non-ionic detergent, on the farnesyl transferase or geranylgeranyl transferase activities were observed with all peptide substrates. Additionally, the effect of temperature was investigated and these assays were applied to determine Michaelis–Menten constants ( K m) of the substrates: dansyl-GCVLS (1.8 μM), dansyl-GCVVM (3.2 μM), dansyl-CVIM (3.4 μM) and dansyl-GCVLL (8.4 μM) and FPP (22.6 μM) for FTase activity. Using GGPP as co-substrate, GGTase activity was measured with K m values superior to 50 μM for all the three substrate dansyl-GCVLS, dansyl-GCVVM, or dansyl-CVIM, whereas values of 7.6 and 5.4 μM were calculated for the dansyl-GCVLL sequence and GGPP co-substrate, respectively. IC 50 values of selective prenyl transferase inhibitors, B-581, FTI 276 and GGTI 287 have been measured to 34, 0.8 and 18 nM, respectively, using dansyl-GCVLS as substrate (FTase inhibition). When dansyl-GCVLL is used as substrate (GGTase inhibition) the IC 50 values are 5100, 75 and 5 nM for B-581, FTI 276 and GGTI 287, respectively. Then, this developed method allowed to evaluate the selectivity of all the three inhibitors tested.

Isoelectric focusing analysis of detergent extracted renal 11β-hydroxysteroid dehydrogenase

11β-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) from rat renal cortex microsomes was solubilized using several detergents, the most effective being Zwittergent 3–10 and Triton X-100. The activity ratio oxidation/reduction of the reversible reaction corticosteroneα 11-dehydrocoticosterone varied depending on the detergent used. We attribute this variation to direct effects of different detergents on enzyme kinetics. In contrast, comparable results obtained with liver 11-HSD have been attributed to the possibility of spatially separated 11-oxidase and 11-reductase activities [1]. In order to test whether renal 11-HSD represents a uniform oxidoreductase as generally assumed, or a dual enzyme system as has been recently proposed [1] an attempt was made to characterize 11-HSD solubilized from renal microsomal fractions using isoelectric focusing (IEF). When 11-HSD was extracted with 1% Triton X-100 (= partially solubilized fraction) a heterogenous peak pattern was obtained. In contrast, IEF of 11-HSD extracted with 10% Triton X-100 (=delipidated fraction) resulted in a single peak at about pH 5.9 with both oxidative and reductive activity at practically identical positions within the gels. From this observation we conclude that the degree of detergent solubilization of a membrane bound protein affects its amphoteric properties and that removal of membranous lipids is a prerequisite for the analysis of its behaviour. Since the more delipidated fraction of 11-HSD revealed only one activity peak the data are compatible with the uniform enzyme concept since oxidative and reductive activities of renal cortical 11-HSD could not be separated.

Detergents inhibit exocytosis in PC 12 cells: evidence for an effect on ion fluxes.

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+-stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X-100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 microM and 10 microM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4). The inhibitory effect of 30 microM Triton X-100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X-100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X-100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X-100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13-14 degrees C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished. In the presence of 30 microM Triton X-100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50-60%. These data suggest that the site of action of Triton X-100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.

Biochemical studies on acid phosphatase in human placenta and fetal liver during development.

Activity and a few physiochemical properties of acid phosphatase (ACP) have been studied in different subcellular fractions of developing human placenta and fetal liver. The enzyme activity is discernible in all subfractions of each tissue already in the 6th week of gestation. The activity of the hydrolase in 20,000 g pellet, 105,000 g pellet and in 105,000 g supernatant of the placenta and in all fractions of the liver increases significantly with advancement of pregnancy until 26 weeks and declines in term placenta. However, 800 and 10,000 g pellets of the placenta show a gradual decrease in ACP activity with progression of gestation. Experiment with different inhibitors reveals that each tissue contains at least two types of ACP--a soluble one and a sedimentable one. It appears from the Lineweaver-Burk plot of kinetic study that sedimentable form has low Km and high Vmax values. Ag+, Cu++, Hg++, Co++ and Zn++ are inhibitory, while Fe++ and EDTA are stimulatory to the enzyme. The effect of different detergents, particularly with sodium deoxycholate and Triton X-100, shows the increase of the hydrolase activity, indicating 'latent nature' of the enzyme.

The Effect of Detergents on the Felting of Wool

. Synthetic anionic and nononic detergents can produce higher felting rates than soap and the concentrations of detergents giving maximum felting rates can vary, with the type of machine used. In rotating-drum machines, maximum felting with synthetic detergents is found at about 0.01% concentration or less, compared with 0.05% in ma chines in which the wool is completely immersed for the duration of the test. With soap, maximum felting occurs at 0.05% in both types of machines. The felting rates with commercial detergent formulations depend mostly on the con centration of active ingredient, although the relatively high pH (>10.0) of some "heavy-duty" detergent solutions may give lower felting rates than those with solutions of pH 6.8-9.6. Tests for felting shrinkage carried out in the absence of detergents do not necessarily give a true indication of the degree of felting which is possible in machine washing, and standard tests may need to be related to the maximum, felting rates which could be encountered in practice. The relative effects of different detergents are more marked with shrink-resistant than untreated wool and wool treated with some processes is more sensitive to the lubricating effect of detergents than that treated with other processes.