-
Protein Science : a Publication of the... Mar 2022Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the...
Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and apply it to screen 61 eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteins (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functionally competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.
Topics: Cell-Free System; Detergents; Eukaryota; High-Throughput Screening Assays; Lipids; Membrane Proteins; Membranes
PubMed: 34910339
DOI: 10.1002/pro.4259 -
Journal of Occupational and... Feb 2022This study evaluated the efficacy of detergent-based surface cleaning methods against Murine Hepatitis Virus A59 (MHV) as a surrogate coronavirus for SARS-CoV-2. MHV (5%...
This study evaluated the efficacy of detergent-based surface cleaning methods against Murine Hepatitis Virus A59 (MHV) as a surrogate coronavirus for SARS-CoV-2. MHV (5% soil load in culture medium or simulated saliva) was inoculated onto four different high-touch materials [stainless steel (SS), Acrylonitrile Butadiene Styrene plastic (ABS), Formica, seat fabric (SF)]. Immediately and 2-hr post-inoculation, coupons were cleaned (damp wipe wiping) with and without pretreatment with detergent solution or 375 ppm hard water. Results identified that physical removal (no pretreatment) removed >2.3 log MHV on ABS, SS, and Formica when surfaces were cleaned immediately. Pretreatment with detergent or hard water increased effectiveness over wet wiping 2-hr post-inoculation; pretreatment with detergent significantly increased ( ≤ 0.05) removal of MHV in simulated saliva, but not in culture media, over hard water pretreatment (Formica and ABS). Detergent and hard water cleaning methods were ineffective on SF under all conditions. Overall, efficacy of cleaning methods against coronaviruses are material- and matrix-dependent; pre-wetting surfaces with detergent solutions increased efficacy against coronavirus suspended in simulated saliva. This study provides data highlighting the importance of incorporating a pre-wetting step prior to detergent cleaning and can inform cleaning strategies to reducing coronavirus surface transmission.
Topics: Animals; COVID-19; Detergents; Humans; Mice; Murine hepatitis virus; Porosity; SARS-CoV-2
PubMed: 34878351
DOI: 10.1080/15459624.2021.2015075 -
Current Protocols Jun 2022Understanding the mechanisms of membrane protein function is critical for biomedical research and drug discovery as membrane proteins constitute ∼30% of the proteins...
Understanding the mechanisms of membrane protein function is critical for biomedical research and drug discovery as membrane proteins constitute ∼30% of the proteins encoded by the genomes of both lower and higher organisms and are targets for two-thirds of approved drugs worldwide. Significant progress has been made in engineering host expression systems for large-scale production of membrane proteins and in determining their three-dimensional high-resolution structures. Despite these efforts, the study of membrane proteins at the atomic level is challenging due to poor expression and extraction, low yields of functional protein, and the complexity and heterogeneity of source membranes. Structural and spectroscopic studies of any membrane protein require that the protein be extracted from its native membranes into a membrane-mimetic stable environment, which is often achieved by the use of detergents. Unfortunately, there is no magic detergent that can extract all membrane proteins and successful extraction often requires a thorough screen of detergents. Furthermore, membrane protein purification in general and the detergents used are very expensive, which puts a financial constraint on sophisticated membrane protein studies. To overcome this hurdle, a dual-detergent strategy has recently been developed and successfully applied to purify various classes of pure, stable, and functionally relevant membrane proteins in a cost-effective manner. This strategy uses an inexpensive detergent for solubilization of the desired protein from membranes and a second detergent during protein purification. In the Basic Protocol, we describe the dual-detergent strategy to significantly reduce the overall purification cost of a bacterial membrane protein using the magnesium ion channel MgtE as an example. Support Protocols are also provided for selecting a suitable E. coli strain for protein expression and the optimal detergent(s) for membrane protein solubilization. © 2022 Wiley Periodicals LLC. Basic Protocol: Expression, membrane solubilization, and cost-effective purification of MgtE Support Protocol 1: Selecting a suitable E. coli strain for optimal protein expression Support Protocol 2: Identification of suitable detergents for membrane protein solubilization.
Topics: Bacterial Proteins; Cost-Benefit Analysis; Detergents; Escherichia coli; Membrane Proteins
PubMed: 35714356
DOI: 10.1002/cpz1.452 -
Biophysical Journal May 2023Single-particle electron cryo-microscopy (cryo-EM) has become an effective and straightforward approach to determine the structure of membrane proteins. However,...
Single-particle electron cryo-microscopy (cryo-EM) has become an effective and straightforward approach to determine the structure of membrane proteins. However, obtaining cryo-EM grids of sufficient quality for high-resolution structural analysis remains a major bottleneck. One of the difficulties arises from the presence of detergents, which often leads to a lack of control of the ice thickness. Amphipathic polymers such as amphipols (APols) are detergent substitutes, which have proven to be valuable tools for cryo-EM studies. In this work, we investigate the physico-chemical behavior of APol- and detergent-containing solutions and show a correlation with the properties of vitreous thin films in cryo-EM grids. This study provides new insight on the potential of APols, allowing a better control of ice thickness while limiting protein adsorption at the air-water interface, as shown with the full-length mouse serotonin 5-HT receptor whose structure has been solved in APol. These findings may speed up the process of grid optimization to obtain high-resolution structures of membrane proteins.
Topics: Animals; Mice; Surface-Active Agents; Detergents; Cryoelectron Microscopy; Electrons; Ice; Membrane Proteins
PubMed: 37077048
DOI: 10.1016/j.bpj.2023.04.016 -
Environmental Science and Pollution... Mar 2023The increasing consumption of cleaning products deteriorates water resources due to harmful components such as phosphorus (P) and nitrogen (N) compounds, oils, bleach,... (Comparative Study)
Comparative Study
The increasing consumption of cleaning products deteriorates water resources due to harmful components such as phosphorus (P) and nitrogen (N) compounds, oils, bleach, and acids, typical compounds in traditional detergents. The use of biodegradable detergents as an environmentally friendly alternative has been proposed in different regions. In Colombia, resolution 1770/2018 sets a minimum biodegradability rate of 60% for the surfactants present in liquid detergents, which would reduce to a similar extent the impacts on water after their use. However, the environmental impacts of the supply chain of these detergents and their raw materials have not been evaluated so far. This study presents an environmental life cycle assessment of petroleum-based liquid detergents and a comparison to traditional solid detergents, based on the ISO 14040 standard and the ReCiPe-2016 impacts assessment method. A novel bio-detergent containing anionic plant-based surfactants was proposed in this analysis. The impacts of packaging and the distribution of the product to consumers were also considered. Raw materials contributed to 91% of the total of 314 g of CO eq generated per liter of liquid detergent, where the production of fatty alcohol sulfate and PET packaging shared 78.8% and 12.2% of the total impact, respectively. It was also determined that 5.4 L of water are consumed and 0.09 g of P eq and 0.1 g of N eq are emitted per liter of detergent. This liquid detergent presented better environmental performance than traditional detergents in all the impact categories, except for the fossil resource scarcity category. The evaluated detergent would significantly mitigate the generation of negative effects on ecosystems. Moreover, the substitution of PET for HDPE packaging could reduce the impacts on freshwater eutrophication by 10%, although the carbon footprint can slightly increase, which could be compensated due to its higher recyclability rate. In contrast, the proposed bio-detergent would not have significant benefits and would negatively affect water consumption and land use in its supply chain.
Topics: Surface-Active Agents; Detergents; Colombia; Plants; Petroleum; Time Factors; Product Packaging; Environment
PubMed: 36508092
DOI: 10.1007/s11356-022-24439-x -
The Journal of Physical Chemistry. B Jun 2015Several membrane proteins and numerous membrane-active peptides have been studied in detergent micelles by solution NMR. However, the detailed structure of these...
Several membrane proteins and numerous membrane-active peptides have been studied in detergent micelles by solution NMR. However, the detailed structure of these complexes remains unknown. We propose a modeling approach that treats the protein and detergent in atomistic detail and the solvent implicitly. The model is based on previous work on dodecylphosphocholine micelles, adapted for use with the CHARMM36 force field and extended to sodium dodecyl sulfate micelles. Solvation parameters were slightly adjusted to reproduce experimental data on aggregation numbers and critical micelle concentrations. To test the approach, several membrane-active peptides and three β-barrel membrane proteins were subjected to molecular dynamics simulations in the presence of a large number of detergent molecules. Their experimentally determined secondary structure was maintained and the RMSD values were less than 2 Å. Deformations were commonly observed in the N or C termini. The atomistic view of the protein-micelle systems that this approach provides could be useful in interpreting biophysical experiments carried out in the presence of detergent.
Topics: Detergents; Micelles; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Phosphorylcholine; Protein Multimerization; Protein Structure, Secondary; Proteins; Sodium Dodecyl Sulfate; Solvents; Water
PubMed: 26035001
DOI: 10.1021/acs.jpcb.5b00171 -
Journal of Applied Microbiology Feb 2022Contaminated laundry can spread infections. However, current directives for safe laundering are limited to healthcare settings and not reflective of domestic conditions....
AIMS
Contaminated laundry can spread infections. However, current directives for safe laundering are limited to healthcare settings and not reflective of domestic conditions. We aimed to use quantitative microbial risk assessment to evaluate household laundering practices (e.g., detergent selection, washing and drying temperatures, and sanitizer use) relative to log reductions in pathogens and infection risks during the clothes sorting, washer/dryer loading, folding and storing steps.
METHODS AND RESULTS
Using published data, we characterized laundry infection risks for respiratory and enteric pathogens relative to a single user contact scenario and a 1.0 × 10 acceptable risk threshold. For respiratory pathogens, risks following cold water wash temperatures (e.g. median 14.4℃) and standard detergents ranged from 2.2 × 10 to 2.2 × 10 . Use of advanced, enzymatic detergents reduced risks to 8.6 × 10 and 2.2 × 10 respectively. For enteric pathogens, however, hot water, advanced detergents, sanitizing agents and drying are needed to reach risk targets.
SIGNIFICANCE AND IMPACT OF THE STUDY
Conclusions provide guidance for household laundry practices to achieve targeted risk reductions, given a single user contact scenario. A key finding was that hand hygiene implemented at critical control points in the laundering process was the most significant driver of infection prevention, additionally reducing infection risks by up to 6 log .
Topics: Detergents; Laundering; Textiles
PubMed: 34465009
DOI: 10.1111/jam.15273 -
Methods in Molecular Biology (Clifton,... 2021Lipid rafts are microdomains on plasma membrane that contain high levels of cholesterol and sphingolipids. Because of the detergent-resistant property of lipid rafts,...
Lipid rafts are microdomains on plasma membrane that contain high levels of cholesterol and sphingolipids. Because of the detergent-resistant property of lipid rafts, lipid rafts isolated by methods that use detergents frequently yield different results. Artifacts can also be introduced through the use of detergents. These limitations could be overcome with a detergent-free method which eliminates possible artificial influences. Importantly, lipid rafts prepared with a detergent-free method is more compatible to mass spectrometric analysis since the ion suppression effect is largely reduced.This chapter describes a detergent-free two-step method for preparation of lipid rafts. Firstly, a purified plasma membrane fraction is prepared from cells by sedimentation of the postnuclear supernatant (PNS) in a Percoll gradient. Secondly, the as-prepared plasma membranes are sonicated to release lipid rafts which are further isolated by flotation in a continuous gradient of Optiprep solution. Then, we introduce a typical shotgun lipidomics workflow that can be used as a cost-effective and relatively high throughput method to determine the lipidomes of lipid rafts.The method also makes an easy start for lipidomics studies.
Topics: Cell Fractionation; Cholesterol; Detergents; Lipidomics; Mass Spectrometry; Membrane Microdomains; Sphingolipids
PubMed: 32770499
DOI: 10.1007/978-1-0716-0814-2_2 -
Biochemistry Jun 2020The structural and functional properties of G protein-coupled receptors (GPCRs) are often studied in a detergent micellar environment, but many GPCRs tend to denature or...
The structural and functional properties of G protein-coupled receptors (GPCRs) are often studied in a detergent micellar environment, but many GPCRs tend to denature or aggregate in short alkyl chain detergents. In our previous work [Lee, S., et al. (2016) , 15425-15433], we showed that GPCRs in alkyl glucosides were highly dynamic, resulting in the penetration of detergent molecules between transmembrane α-helices, which is the initial step in receptor denaturation. Although this was not observed for GPCRs in dodecyl maltoside (DDM, also known as lauryl maltoside), even this detergent is not mild enough to preserve the integrity of many GPCRs during purification. Lauryl maltose neopentylglycol (LMNG) detergents have been found to have significant advantages for purifying GPCRs in a native state as they impart more stability to the receptor than DDM. To gain insights into how they stabilize GPCRs, we used atomistic molecular dynamics simulations of wild type adenosine A receptor (WT-AR), thermostabilized AR (tAR), and wild type β-adrenoceptor (βAR) in a variety of detergents (LMNG, DMNG, OGNG, and DDM). Analysis of molecular dynamics simulations of tAR in LMNG, DMNG, and OGNG showed that this series of detergents exhibited behavior very similar to that of an analogous series of detergents DDM, DM, and OG in our previous study. However, there was a striking difference upon comparison of the behavior of LMNG to that of DDM. LMNG showed considerably less motion than DDM, which resulted in the enhanced density of the aliphatic chains around the hydrophobic regions of the receptor and considerably more hydrogen bond formation between the head groups. This contributed to enhanced interaction energies between both detergent molecules and between the receptor and detergent, explaining the enhanced stability of GPCRs purified in this detergent. Branched detergents occlude between transmembrane helices and reduce their flexibility. Our results provide a rational foundation to develop detergent variants for stabilizing membrane proteins.
Topics: Detergents; HEK293 Cells; Humans; Micelles; Molecular Dynamics Simulation; Molecular Structure; Protein Stability; Receptors, G-Protein-Coupled
PubMed: 32437610
DOI: 10.1021/acs.biochem.0c00183 -
Nature Communications Apr 2021Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized...
Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these complexes remains challenging because of their chemical composition, the hydrophobicity and structural instability of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid supramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework (ZIF) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF bio-composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embedded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization.
Topics: Cell Membrane; Copper-Transporting ATPases; Detergents; Escherichia coli Proteins; Exoskeleton Device; Immobilization; Kinetics; Lipid Bilayers; Membrane Proteins; Micelles; Phospholipids; Proteolipids; Scattering, Radiation; Unilamellar Liposomes; X-Ray Diffraction
PubMed: 33850135
DOI: 10.1038/s41467-021-22285-y