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Biophysical Journal Jul 2013Multiple data are available on the self-assembly of mixtures of bilayer-forming amphiphiles, particularly phospholipids and micelle-forming amphiphiles, commonly denoted... (Review)
Review
Multiple data are available on the self-assembly of mixtures of bilayer-forming amphiphiles, particularly phospholipids and micelle-forming amphiphiles, commonly denoted detergents. The structure of such mixed assemblies has been thoroughly investigated, described in phase diagrams, and theoretically rationalized in terms of the balance between the large spontaneous curvature of the curvophilic detergent and the curvophobic phospholipids. In this critical review, we discuss the mechanism of this process and try to explain the actual mechanism involved in solubilization. Interestingly, membrane solubilization by some detergents is relatively slow and the common attribute of these detergents is that their trans-bilayer movement, commonly denoted flip-flop, is very slow. Only detergents that can flip into the inner monolayer cause relatively rapid solubilization of detergent-saturated bilayers. This occurs via the following sequence of events: 1), relatively rapid penetration of detergent monomers into the outer monolayer; 2), trans-membrane equilibration of detergent monomers between the two monolayers; 3), saturation of the bilayer by detergents and consequent permeabilization of the membrane; and 4), transition of the whole bilayer to thread-like mixed micelles. When the detergent cannot flip to the inner monolayer, the outer monolayer becomes unstable due to mass imbalance between the monolayers and inclusion of the curvophilic detergent molecules in a flat surface. Consequently, the outer monolayer forms mixed micellar structures within the outer monolayer. Shedding of these micelles into the aqueous solution results in partial solubilization. The consequent leakage of detergent into the liposome results in trans-membrane equilibration of detergent and subsequent micellization through the rapid bilayer-saturation mechanism.
Topics: Detergents; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Liposomes; Micelles; Solubility
PubMed: 23870250
DOI: 10.1016/j.bpj.2013.06.007 -
Biochimica Et Biophysica Acta Nov 2000The process of formation of lipid vesicles using the technique of detergent removal from mixed-micelles is examined. Recent studies on the solubilization and... (Comparative Study)
Comparative Study Review
The process of formation of lipid vesicles using the technique of detergent removal from mixed-micelles is examined. Recent studies on the solubilization and reconstitution of liposomes participated to our knowledge of the structure and properties of mixed lipid-detergent systems. The mechanisms involved in both the lipid self assembly and the micelle-vesicle transition are first reviewed. The simplistic three step minimum scheme is described and criticized in relation with isothermal as well as a function of the [det]/[lip] ratio, phase diagram explorations. The techniques of detergent elimination are reviewed and criticized for advantages and disadvantages. New methods inducing micelle-vesicle transition using enzymatic reaction and T-jump are also described and compared to more classical ones. Future developments of these techniques and improvements resulting of their combinations are also considered. Proper reconstitution of membrane constituents such as proteins and drugs into liposomes are examined in the light of our actual understanding of the micelle-vesicle transition.
Topics: Detergents; Electrolytes; Fats; Lipids; Liposomes; Methods; Micelles; Models, Molecular; Particle Size; Phospholipids; Water
PubMed: 11090817
DOI: 10.1016/s0304-4157(00)00006-x -
Journal of Microbiological Methods Dec 2023Leishmaniasis is an infectious disease caused by protozoan species in the genera Leishmania and Endotrypanum. Current antileishmanial drugs are limited due to adverse...
Leishmaniasis is an infectious disease caused by protozoan species in the genera Leishmania and Endotrypanum. Current antileishmanial drugs are limited due to adverse effects, variable efficacy, the development of resistant parasites, high cost, parenteral administration and lack of availability in endemic areas. Therefore, active searching for new antileishmanial drugs has been done for years, mainly by academia. Drug screening techniques have been a challenge since the intracellular localization of Leishmania amastigotes implies that the host cell may interfere with the quantification of the parasites and the final estimation of the effect. One of the procedures to avoid host cell interference is based on its detergent-mediated lysis and subsequent transformation of viable amastigotes into promastigotes, their proliferation and eventual quantification as an axenic culture of promastigotes. However, the use of detergent involves additional handling of cultures and variability. In the present work, cultures of intracellular amastigotes were incubated for 72 h at 26 °C after exposure to the test compounds and the transformation and proliferation of parasites took place without need of adding any detergent. The assay demonstrated clear differentiation of negative and positive controls (average Z´ = 0.75) and 50% inhibitory concentrations of compounds tested by this method and by the gold standard enumeration of Giemsa-stained cultures were similar (p = 0.5002) and highly correlated (r = 0.9707). This simplified procedure is less labor intensive, the probability of contamination and the experimental error are reduced, and it is appropriate for the automated high throughput screening of compounds.
Topics: Animals; Leishmania; Parasites; Drug Evaluation, Preclinical; Detergents; Leishmaniasis; Antiprotozoal Agents
PubMed: 37871728
DOI: 10.1016/j.mimet.2023.106847 -
Journal of Materials Science. Materials... May 2022Detergent treatment is the most commonly used method for the decellularization of ligaments and tendon grafts. However, it is well recognized that detergent treatment...
Detergent treatment is the most commonly used method for the decellularization of ligaments and tendon grafts. However, it is well recognized that detergent treatment can also adversely affect the extracellular matrix. This study found that discission into the aponeurosis layer of the patellar tendon (PT) before decellularization is conducive to extracting cells from the PT using a low quantity of detergent in a short time period. The acellular aponeurosis discission ligament (AADL) retains its native collagen fibril structure and mechanical properties. Moreover, the PT retained cell and tissue compatibility in vitro and in vivo. After implantation into a defective allogeneic PT, we found that the AADL healed well in the host, and its collagen structure exhibited gradual improvement 12 months after implantation with satisfactory reconstruction. IMPACT: The aponeurosis of tendons/ligaments is the main barrier to achieving complete decellularization, and it thus prevents complete recellularization for applications in tissue engineering. Aponeurosis can obstruct the removal of cell components. We found that excising the aponeurosis before decellularization allows for the removal of cellular components with a reduced amount of detergent, thus improving the biological properties of the acellular ligament. To the best of our knowledge, no similar studies have been performed. Graphical abstract.
Topics: Aponeurosis; Collagen; Detergents; Extracellular Matrix; Ligaments; Tissue Engineering; Tissue Scaffolds
PubMed: 35507049
DOI: 10.1007/s10856-022-06661-8 -
The Medical Journal of Australia Apr 1954
Topics: Aerosols; Cough; Detergents; Humans; Respiratory Therapy
PubMed: 13164649
DOI: 10.5694/j.1326-5377.1954.tb85936.x -
Scientific Reports Jul 2019Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to...
Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.
Topics: Detergents; Fluorometry; High-Throughput Screening Assays; Membrane Proteins; Protein Stability; Solubility
PubMed: 31316088
DOI: 10.1038/s41598-019-46686-8 -
Cold Spring Harbor Protocols Dec 2017This protocol describes the lysis of tissue culture cells for the solubilization of proteins of interest for immunoprecipitation. Upon collection of cells by...
This protocol describes the lysis of tissue culture cells for the solubilization of proteins of interest for immunoprecipitation. Upon collection of cells by centrifugation and depending on the use of either Tris- or phosphate-based cell lysis buffers, cells are rinsed, respectively, in either TBS or PBS before lysis. If possible, the pH of the Wash buffer should match that of the Lysis buffer. Adherent cells can be directly lysed on the plate. This is particularly useful upon lysis encompassing mild nonionic detergents, leaving the cytoskeleton intact. Alternatively, adherent cells can be scraped off the plate and directly resuspended in Lysis buffer or Wash buffer for transfer to a tube followed by the addition of Lysis buffer.
Topics: Buffers; Complex Mixtures; Cytological Techniques; Detergents; Hydrogen-Ion Concentration; Immunoprecipitation
PubMed: 29196599
DOI: 10.1101/pdb.prot098558 -
Methods in Molecular Biology (Clifton,... 2022Enrichment of detergent insoluble proteins is a commonly used technique for analyzing proteins that may be aggregating in disease or with age. However, various methods...
Enrichment of detergent insoluble proteins is a commonly used technique for analyzing proteins that may be aggregating in disease or with age. However, various methods for enriching for these proteins are used. Here we present a method using a mild detergent (Triton X-100) and high centrifugation speed (20,000 × g) allowing for sufficient protein extraction and enrichment for large protein assemblies. Digestion is performed on columns allowing for a methanol chloroform wash to remove the highly prevalent lipids in brain tissue. This is followed by analysis by data independent acquisition mass spectrometry, which we have found to be highly reproducible. Our method is intended to enrich for amorphous aggregates, which may accumulate upon the collapse of protein homeostasis.
Topics: Animals; Brain; Detergents; Mass Spectrometry; Mice; Octoxynol; Proteins
PubMed: 35612733
DOI: 10.1007/978-1-0716-2124-0_3 -
Angewandte Chemie (International Ed. in... Apr 2015Surfactants carrying fluorocarbon chains hold great promise as gentle alternatives to conventional hydrocarbon-based detergents for the solubilization and handling of...
Surfactants carrying fluorocarbon chains hold great promise as gentle alternatives to conventional hydrocarbon-based detergents for the solubilization and handling of integral membrane proteins. However, their inertness towards lipid bilayer membranes has limited the usefulness of fluorinated surfactants in situations where detergent-like activity is required. We demonstrate that fluorination does not necessarily preclude detergency, as exemplified by a fluorinated octyl maltoside derivative termed F6 OM. This nonionic compound readily interacts with and completely solubilizes phospholipid vesicles in a manner reminiscent of conventional detergents without, however, compromising membrane order at subsolubilizing concentrations. Owing to this mild and unusual mode of detergency, F6 OM outperforms a lipophobic fluorinated surfactant in chaperoning the functional refolding of an integral membrane enzyme by promoting bilayer insertion in the absence of micelles.
Topics: Detergents; Halogenation; Liposomes; Membrane Proteins; Micelles; Phosphatidylcholines; Protein Refolding; Surface-Active Agents
PubMed: 25753129
DOI: 10.1002/anie.201412359 -
Journal of the American Society For... Dec 2023Membrane proteins (MPs) play many critical roles in cellular physiology and constitute the majority of current pharmaceutical targets. However, MPs are comparatively...
Membrane proteins (MPs) play many critical roles in cellular physiology and constitute the majority of current pharmaceutical targets. However, MPs are comparatively understudied relative to soluble proteins due to the challenges associated with their solubilization in membrane mimetics. Native mass spectrometry (nMS) has emerged as a useful technique to probe the structures of MPs. Typically, nMS studies using MPs have employed detergent micelles to solubilize the MP. Oftentimes, the detergent micelle that the MP was purified in will be exchanged into another detergent prior to analysis by nMS. While methodologies for performing detergent exchange have been extensively described in prior reports, the effectiveness of these protocols remains understudied. Here, we present a critical analysis of detergent exchange efficacy using several model transmembrane proteins and a variety of commonly used detergents, evaluating the completeness of the exchange using a battery of existing protocols. Our data include results for octyl glucoside (OG), octaethylene glycol monododecyl ether (C12E8), and tetraethylene glycol monooctyl ether (C8E4), and these data demonstrate that existing protocols are insufficient and yield incomplete exchange for the proteins under the conditions probed here. In some cases, our data indicate that up to 99% of the measured detergent corresponds to the original pre-exchange detergent rather than the desired post-exchange detergent. We conclude by discussing the need for new detergent exchange methodologies alongside improved exchange yield expectations for studying the potential influence of detergents on MP structures.
Topics: Membrane Proteins; Detergents; Micelles; Mass Spectrometry; Ethers
PubMed: 37956121
DOI: 10.1021/jasms.3c00230