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The Journal of Physical Chemistry... Apr 2018Gradual dissociation of detergent molecules from water-insoluble membrane proteins culminates in protein aggregation. However, the time-dependent trajectory of this...
Gradual dissociation of detergent molecules from water-insoluble membrane proteins culminates in protein aggregation. However, the time-dependent trajectory of this process remains ambiguous because the signal-to-noise ratio of most spectroscopic and calorimetric techniques is drastically declined by the presence of protein aggregates in solution. We show that by using steady-state fluorescence polarization (FP) spectroscopy the dissociation of the protein-detergent complex (PDC) can be inspected in real time at detergent concentrations below the critical micelle concentration. This article provides experimental evidence of the coexistence of two distinct phases of the dissociations of detergent monomers from membrane proteins. We first noted a slow detergent predesolvation process, which was accompanied by a relatively modest change in the FP anisotropy, suggesting a small number of dissociated detergent monomers from the proteomicelles. This predesolvation phase was followed by a fast detergent desolvation process, which was highlighted by a major alteration in the FP anisotropy. The durations and rates of these phases were dependent on both the detergent concentration and the interfacial PDC interactions. Further development of this approach might lead to the creation of a new semiquantitative method for the assessment of the kinetics of association and dissociation of proteomicelles.
Topics: Bacterial Outer Membrane Proteins; Detergents; Fluorescence Polarization; Kinetics
PubMed: 29595981
DOI: 10.1021/acs.jpclett.8b00549 -
Journal of Pharmaceutical and... Sep 2022Membrane proteins constitute around 20-30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets....
Membrane proteins constitute around 20-30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of "omics" techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.
Topics: Animals; Detergents; Electrophoresis, Polyacrylamide Gel; Humans; Hydrophobic and Hydrophilic Interactions; Mammals; Membrane Proteins; Mice; Octoxynol
PubMed: 35839578
DOI: 10.1016/j.jpba.2022.114926 -
Langmuir : the ACS Journal of Surfaces... Mar 2022In the decades'-long quest for high-quality membrane protein (MP) crystals, non-ionic detergent micelles have primarily served as a passive shield against protein...
In the decades'-long quest for high-quality membrane protein (MP) crystals, non-ionic detergent micelles have primarily served as a passive shield against protein aggregation in aqueous solution and/or as a conformation stabilizing environment. We have focused on exploiting the physical chemistry of detergent micelles in order to direct intrinsic MP/detergent complexes to assemble via conjugation under ambient conditions, thereby permitting finely tuned control over the micelle cloud point. In the current work, three commercially available amphiphilic, bipyridine chelators in combination with Fe or Ni were tested for their ability to conjugate non-ionic detergent micelles both in the presence and absence of an encapsulated bacteriorhodopsin molecule. Water-soluble chelators were added, and results were monitored with light microscopy and dynamic light scattering (DLS). [Bipyridine:metal] complexes produced micellar conjugates, which appeared as oil-rich globules (10-200 μm) under a light microscope. DLS analysis demonstrated that micellar conjugation is complete 20 min after the introduction of the amphiphilic complex, and that the conjugation process can be fully or partially reversed with water-soluble chelators. This process of controlled conjugation/deconjugation under nondenaturing conditions provides broader flexibility in the choice of detergent for intrinsic MP purification and conformational flexibility during the crystallization procedure.
Topics: Bacteriorhodopsins; Crystallization; Detergents; Micelles; Water
PubMed: 35179381
DOI: 10.1021/acs.langmuir.1c03343 -
Journal of Visualized Experiments : JoVE Mar 2018Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in...
Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.
Topics: Animals; Carrier Proteins; Detergents; Humans; Membrane Proteins; Protein Transport
PubMed: 29578521
DOI: 10.3791/57179 -
The Journal of Physical Chemistry. B Nov 2017Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly...
Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semiquantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.
Topics: Detergents; Fluorescence Polarization; Hydrophobic and Hydrophilic Interactions; Membrane Proteins; Molecular Dynamics Simulation
PubMed: 29035562
DOI: 10.1021/acs.jpcb.7b08045 -
Cells, Tissues, Organs 2023Decellularized scaffolds applied in tissue engineering offer improvements, supplying the elevated necessity for organs and tissues for replacement. However, obtaining a...
Decellularized scaffolds applied in tissue engineering offer improvements, supplying the elevated necessity for organs and tissues for replacement. However, obtaining a functional trachea for autotransplantation or allotransplantation is tricky due to the organ anatomical and structural complexity. Most tracheal decellularization protocols are lengthy, expensive, and could damage the tracheal extracellular matrix (ECM) architecture and functionality. Here, we aimed to evaluate the effectiveness of 3 different decellularization protocols combined with chemical and physical methods to obtain acellular canine tracheal scaffolds. Six adult dog tracheas were incised (tracheal segments) resulting in 28 rings for control tissue and 84 rings for decellularization (5-7 mm thick). Subsequently, decellularized tracheal scaffolds were microscopically/macroscopically characterized by histological analysis (Hematoxylin-Eosin, Masson's trichrome, Picrosirius red, Alcian blue, and Safranin O), immunohistochemistry for ECM components, scanning electron microscopy, and genomic DNA quantification. After decellularization, the tracheal tissue revealed reduced genomic DNA, and maintenance of ECM components preserved (structural proteins, adhesive glycoproteins, glycosaminoglycans and proteoglycans), suggesting ECM integrity and functionality. Comparatively, the combined ionic detergent with high vacuum pressure decellularization protocol revealed superior genomic DNA decrease (13.5 ng/mg) and improvement on glycosaminoglycans and proteoglycans preservation regarding the other decellularized trachea scaffolds and native tissue. Our results indicate that the 3 chemical/physical protocols reduce the decellularization time without ECM proteins damage. Notwithstanding, the use of ionic detergent under vacuum pressure was able to generate an innovative strategy to obtain acellular canine tracheal scaffolds with the highest levels of adhesive proteins that support its potentiality for recellularization and future tissue engineering application.
Topics: Dogs; Animals; Tissue Scaffolds; Trachea; Detergents; Vacuum; Tissue Engineering; Extracellular Matrix; Proteoglycans; Glycosaminoglycans; DNA
PubMed: 35640555
DOI: 10.1159/000525273 -
Cold Spring Harbor Protocols Apr 2010Although SDS-PAGE is the method of choice for most denaturing gel electrophoresis procedures, the anionic detergent SDS still presents some drawbacks. For example, SDS...
Although SDS-PAGE is the method of choice for most denaturing gel electrophoresis procedures, the anionic detergent SDS still presents some drawbacks. For example, SDS forms crystals at low temperatures and, in some cases, causes proteins to aggregate or precipitate. In addition, some proteins are not well-resolved in SDS gels or may migrate anomalously. In these situations, the use of a cationic detergent for PAGE offers an alternative approach. The system described in this protocol uses the cationic detergent cetyltrimethyl ammonium bromide (CTAB) and includes a stacking gel based on the zwitterion arginine (used as a stacking agent) and tricine (N-tris[hydroxymethyl]-methylglycine) used as a counterion and buffer. Some proteins separated on the CTAB electrophoresis system retain their native enzymatic activity, provided the samples are prepared without boiling and without the addition of a reducing agent.
Topics: Cetrimonium; Cetrimonium Compounds; Detergents; Electrophoresis, Polyacrylamide Gel; Proteins
PubMed: 20360366
DOI: 10.1101/pdb.prot5412 -
The Journal of Hospital Infection Oct 2023Transmission of infections via contaminated endoscopes is a common problem. Manual cleaning, using at least a detergent, is an important step in endoscope processing and...
BACKGROUND
Transmission of infections via contaminated endoscopes is a common problem. Manual cleaning, using at least a detergent, is an important step in endoscope processing and should be performed as soon as possible to avoid drying of organic residues that might interfere with high-level disinfection and promote biofilm formation.
AIM
To assess the efficacy of two detergent-disinfectants, enzymatic and non-enzymatic, and of an enzymatic detergent used during the manual cleaning against a Klebsiella pneumoniae biofilm.
METHODS
A 24 h biofilm statically formed in a Tygon tube was exposed to detergent-disinfectants at 20 °C and 35 °C for 10 mn, and to enzymatic detergent at 45 °C for 60 mn. The logarithmic reduction in bacteria in the Tygon tube and the number of bacteria in the product supernatant were calculated.
FINDINGS
Biofilm formation was reproducible between assays. After exposure to detergent-disinfectants, the logarithmic reduction was between 6.32 and 6.71 log cfu/cm in the Tygon tubes. No bacteria were found in their supernatants. Results in the detergent-disinfectant group were not affected by the exposure temperature or the addition of enzymes. No decrease in the bacterial load was observed in the Tygon tubes after exposure to the enzymatic detergent. Bacteria were found in its supernatant.
CONCLUSION
These results show the importance of the choice of products used during the manual cleaning phase. They also show the potential benefit of combining detergent and disinfectant activity to decrease the bacterial load during the manual cleaning step of endoscope processing.
Topics: Humans; Disinfectants; Klebsiella pneumoniae; Detergents; Disinfection; Endoscopes; Biofilms
PubMed: 37487794
DOI: 10.1016/j.jhin.2023.04.019 -
Pediatrics Sep 1989After ingesting or inhaling laundry detergent powder, eight children required hospital admission. The predominant symptoms were stridor, drooling, and respiratory... (Review)
Review
After ingesting or inhaling laundry detergent powder, eight children required hospital admission. The predominant symptoms were stridor, drooling, and respiratory distress. All but one patient underwent endoscopy of the airways and the esophagus, five children were admitted to the intensive care unit, and four children required endotracheal intubation. Laundry detergent ingestions are generally considered to have minor consequences, and there exists a paucity of literature on the subject. Evidence of significant morbidity incurred because of ingestion or inhalation of sodium carbonate-containing laundry detergent powder is presented, together with a review of the existing literature.
Topics: Burns, Chemical; Carbonates; Child, Preschool; Detergents; Female; Household Products; Humans; Infant; Laundering; Male; Powders; Respiration Disorders; Surface-Active Agents; Vomiting
PubMed: 2671913
DOI: No ID Found -
The Journal of Physical Chemistry. B Feb 2017Detergents are commonly applied in lipase assays to solubilize sparingly soluble model substrates. However, detergents affect lipases as well as substrates in multiple...
Detergents are commonly applied in lipase assays to solubilize sparingly soluble model substrates. However, detergents affect lipases as well as substrates in multiple ways. The effect of detergents on lipase activity is commonly attributed to conformational changes in the lid region. This study deals with the effect of the nonionic detergent, poly(ethylene glycol) dodecyl ether, on a lipase that does not contain a lid sequence, lipase A from Bacillus subtilis (BSLA). We show that BSLA activity depends strongly on the detergent concentration and the dependency profile changes with pH. The interaction of BSLA with detergent monomers and micelles is studied using fluorescence correlation spectroscopy, time-resolved anisotropy decay, and temperature-induced unfolding. Detergent-dependent hydrolysis kinetics of two different substrates at two pH values are fitted with a microkinetic model. This analysis shows that the mechanism of interfacial lipase catalysis is strongly affected by the detergent. It reveals an activation mechanism by monomeric detergent that does not result from structural changes of the lipase. Instead, we propose that interfacial diffusion of the lipase is enhanced by detergent binding.
Topics: Bacillus subtilis; Detergents; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethers; Kinetics; Lipase; Molecular Structure; Polyethylene Glycols; Structure-Activity Relationship
PubMed: 28106397
DOI: 10.1021/acs.jpcb.6b11037