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Proceedings of the National Academy of... Dec 2018Membrane proteins function in native cell membranes, but extraction into isolated particles is needed for many biochemical and structural analyses. Commonly used...
Membrane proteins function in native cell membranes, but extraction into isolated particles is needed for many biochemical and structural analyses. Commonly used detergent-extraction methods destroy naturally associated lipid bilayers. Here, we devised a detergent-free method for preparing cell-membrane nanoparticles to study the multidrug exporter AcrB, by cryo-EM at 3.2-Å resolution. We discovered a remarkably well-organized lipid-bilayer structure associated with transmembrane domains of the AcrB trimer. This bilayer patch comprises 24 lipid molecules; inner leaflet chains are packed in a hexagonal array, whereas the outer leaflet has highly irregular but ordered packing. Protein side chains interact with both leaflets and participate in the hexagonal pattern. We suggest that the lipid bilayer supports and harmonizes peristaltic motions through AcrB trimers. In AcrB D407A, a putative proton-relay mutant, lipid bilayer buttresses protein interactions lost in crystal structures after detergent-solubilization. Our detergent-free system preserves lipid-protein interactions for visualization and should be broadly applicable.
Topics: Cell Membrane; Crystallography, X-Ray; Detergents; Escherichia coli; Escherichia coli Proteins; Lipid Bilayers; Multidrug Resistance-Associated Proteins; Nanoparticles; Protein Conformation
PubMed: 30509977
DOI: 10.1073/pnas.1812526115 -
Biotechnology and Bioengineering Nov 2021Pathogen surface antigens are at the forefront of the viral strategy when invading host organisms. These antigens, including membrane proteins (MPs), are broadly...
Pathogen surface antigens are at the forefront of the viral strategy when invading host organisms. These antigens, including membrane proteins (MPs), are broadly targeted by the host immune response. Obtaining these MPs in a soluble and stable form constitutes a real challenge, regardless of the application purposes (e.g. quantification/characterization assays, diagnosis, and preventive and curative strategies). A rapid process to obtain a native-like antigen by solubilization of a full-length MP directly from a pathogen is reported herein. Rabies virus (RABV) was used as a model for this demonstration and its full-length G glycoprotein (RABV-G) was stabilized with amphipathic polymers, named amphipols (APols). The stability of RABV-G trapped in APol A8-35 (RABV-G/A8-35) was evaluated under different stress conditions (temperature, agitation, and light exposure). RABV-G/A8-35 in liquid form exhibited higher unfolding temperature (+6°C) than in detergent and was demonstrated to be antigenically stable over 1 month at 5°C and 25°C. Kinetic modeling of antigenicity data predicted antigenic stability of RABV-G/A8-35 in a solution of up to 1 year at 5°C. The RABV-G/A8-35 complex formulated in an optimized buffer composition and subsequently freeze-dried displayed long-term stability for 2-years at 5, 25, and 37°C. This study reports for the first time that a natural full-length MP extracted from a virus, complexed to APols and subsequently freeze-dried, displayed long-term antigenic stability, without requiring storage under refrigerated conditions.
Topics: Antigens, Viral; Detergents; Freeze Drying; Protein Stability; Rabies virus; Viral Envelope Proteins
PubMed: 34297405
DOI: 10.1002/bit.27900 -
The Cochrane Database of Systematic... Jul 2018Problems attributed to the accumulation of wax (cerumen) are among the most common reasons for people to present to their general practitioners with ear trouble.... (Meta-Analysis)
Meta-Analysis Review
BACKGROUND
Problems attributed to the accumulation of wax (cerumen) are among the most common reasons for people to present to their general practitioners with ear trouble. Treatment for this condition often involves use of a wax softening agent (cerumenolytic) to disperse the cerumen, reduce the need for, or facilitate syringing, but there is no consensus on the effectiveness of the variety of cerumenolytics in use.
OBJECTIVES
To assess the effectiveness of ear drops (cerumenolytics) for the removal of symptomatic ear wax.
SEARCH METHODS
We searched the Cochrane Ear, Nose and Throat Disorders Group Trials Register; the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library, 2008 issue 2); MEDLINE; EMBASE; CINAHL; ISI Proceedings; Cambridge Scientific Abstracts; mRCT and additional sources for published and unpublished trials. The date of the most recent search was April 2008.
SELECTION CRITERIA
We identified all randomised controlled trials in which a cerumenolytic was compared with no treatment, a placebo, or other cerumenolytics in participants with obstructing or impacted ear wax, and in which the proportion of participants with sufficient clearance of the external canal to make further mechanical clearance unnecessary (primary outcome measure) was stated or calculable.
DATA COLLECTION AND ANALYSIS
The two authors reviewed all the retrieved trials and applied the inclusion criteria independently.
MAIN RESULTS
Nine trials satisfied the inclusion criteria. In all, 679 participants received one of 11 different cerumenolytics. One trial compared active treatments with no treatment, three compared active treatments with water or a saline 'placebo', and all nine trials compared two or more active treatments. Eight trials included syringing as a secondary intervention.Overall, results were inconclusive. The majority of comparisons showed no difference between treatments. Meta-analysis of two high quality trials produced a statistical difference in favour of triethanolamine polypeptide over saline in preventing the need for syringing, but no other significant differences between treatments.In three trials of high to moderate quality, no difference was found between the effectiveness of either sodium bicarbonate ear drops, chlorbutanol, triethanolamine polypeptide oleate condensate or docusate sodium liquid versus a sterile water or saline 'placebo'.One trial of moderate methodological quality found all three treatments - sodium bicarbonate ear drops, chlorbutanol and sterile water - to be significantly better than no treatment at preventing the need for syringing.None of the higher quality trials demonstrated superiority of one agent over another in direct comparisons.
AUTHORS' CONCLUSIONS
Trials have been heterogeneous and generally of low or moderate quality, making it difficult to offer any definitive recommendations on the effectiveness of cerumenolytics for the removal of symptomatic ear wax. Using drops of any sort appears to be better than no treatment, but it is uncertain if one type of drop is any better than another. Future trials should be of high methodological quality, have large sample sizes, and compare both oil-based and water-based solvents with placebo, no treatment or both.
Topics: Cerumen; Detergents; Humans; Randomized Controlled Trials as Topic; Solvents; Syringes
PubMed: 30040120
DOI: 10.1002/14651858.CD004326.pub3 -
Biochimica Et Biophysica Acta.... Nov 2017The immunity proteins against pore-forming colicins represent a family of integral membrane proteins that reside in the inner membrane of producing cells. Cai, the...
The immunity proteins against pore-forming colicins represent a family of integral membrane proteins that reside in the inner membrane of producing cells. Cai, the colicin A immunity protein, was characterized here in detergent micelles by circular dichroism (CD), size exclusion chromatography, chemical cross-linking, nuclear magnetic resonance (NMR) spectroscopy, cysteine accessibility, and colicin A binding in detergent micelles. Bile-salt derivatives induced extensive protein polymerization that precluded further investigation. The physical characterization of detergent-solubilized protein indicates that phosphate-containing detergents are more efficient in extracting, solubilizing and maintaining Cai in a monomeric state. Yet, their capacity to ensure protein activity, reconstitution, helix packing, and high-quality NMR spectra was inferior to that of milder detergents. Solvent ionic strength and composition greatly modified the solubilizing capacity of milder detergents. Most importantly, binding to the colicin A pore-forming domain (pf-ColA) occurred almost exclusively in sugar-derived detergents. The relative performance of the different detergents in each experiment depends on their impact not only on Cai structure, solubility and oligomerization state, but also on other reaction components and technical aspects. Thus, proteoliposomes were best obtained from protein in LDAO micelles, possibly also due to indirect effects on the lipidic bilayer. The compatibility of a detergent with Cai/pf-ColA complex formation is influenced by its effect on the conformational landscape of each protein, where detergent-mediated pf-ColA denaturation could also lead to negative results. The NMR spectra were greatly affected by the solubility, monodispersity, fold and dynamics of the protein-detergent complexes, and none of those tested here provided NMR spectra of sufficient quality to allow for peak assignment. Cai function could be proven in alkyl glycosides and not in those detergents that afforded the best solubility, reconstitution efficiency or spectral quality indicating that these criteria cannot be taken as unambiguous proof of nativeness without the support of direct activity measurements.
Topics: Amino Acid Sequence; Chromatography, Gel; Circular Dichroism; Colicins; Detergents; Escherichia coli; Lipid Bilayers; Magnetic Resonance Spectroscopy; Micelles; Sequence Analysis, Protein; Solubility
PubMed: 28803731
DOI: 10.1016/j.bbamem.2017.08.007 -
Scientific Reports Feb 2017Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain...
Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.
Topics: Detergents; Liposomes; Membrane Proteins; Micelles; Models, Molecular; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 28176812
DOI: 10.1038/srep41751 -
The International Journal of... May 2022Fluorescence correlation spectroscopy (FCS) is a quantitative spectroscopy technique which could potentially increase throughput and sensitivity of screening for ligand,... (Review)
Review
Fluorescence correlation spectroscopy (FCS) is a quantitative spectroscopy technique which could potentially increase throughput and sensitivity of screening for ligand, substrate and inhibitor binding to membrane proteins in solution. However, the purification of membrane proteins in their active forms is complex, as the lipid bilayer provides stability and its removal often causes the protein to become conformationally unstable. This has limited the application of biophysical techniques such as FCS to study the function of membrane proteins. The recent application of native extraction techniques such as styrene maleic acid lipid particles (SMALPs) has resolved this issue and FCS has emerged as a powerful option for studying proteins extracted in this way. This review will discuss the application of FCS to study purified membrane proteins in detergent micelles, nanodiscs and SMALPs and its potential to be used routinely in membrane protein drug discovery.
Topics: Detergents; Fluorescence; Lipid Bilayers; Membrane Proteins; Polystyrenes
PubMed: 35390493
DOI: 10.1016/j.biocel.2022.106210 -
Biophysical Chemistry May 2023Detergents are valuable tools to extract membrane proteins for biophysical, biochemical, and structural scrutiny. The detergent-driven solubilization of bilayers made...
Detergents are valuable tools to extract membrane proteins for biophysical, biochemical, and structural scrutiny. The detergent-driven solubilization of bilayers made from a single lipid species is commonly described in terms of pseudo-phase diagrams and a three-stage model accounting for three ranges comprising (i) intact vesicles, (ii) vesicle/micelle co-existence, or (iii) mixed micelles. Moreover, the pseudo-phase boundaries thus determined can often be quantitatively rationalized in terms of the molecular shapes of the lipid and the detergent used. Yet, it has remained unclear to what extent this approach can be applied to multi-component lipid membranes that more closely mimic the compositional complexity of cellular membranes. Here, we studied how lipid mixtures composed of palmitoyl oleoyl phosphatidylethanolamine (POPE), palmitoyl oleoyl phosphatidylglycerol (POPG), and tetraoleoyl cardiolipin (TOCL) are solubilized by the commonly used zwitterionic detergent lauryldimethylamine N-oxide using isothermal titration calorimetry. While phase diagrams of the diverse lipid mixtures showed the typical ranges of the three-stage model, we found that POPG-rich POPE/POPG bilayers are more difficult to solubilize than POPG-poor POPE/POPG bilayers. In turn, POPE/POPG/TOCL bilayers became increasingly resistant to detergent with increasing TOCL content. Since POPG is nearly cylindrically shaped and TOCL adopts inverted cone-like shapes under current buffer conditions, our solubilization data do not align with shape-based arguments. Instead, additional electrostatic interactions between lipids and detergents lead to non-additive mixing behavior affecting the resilience of complex lipid bilayers against solubilization.
Topics: Detergents; Lipid Bilayers; Cell Membrane; Cardiolipins; Calorimetry; Micelles
PubMed: 36921495
DOI: 10.1016/j.bpc.2023.107002 -
Molecules (Basel, Switzerland) Apr 2016Nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol) is a naturally occurring sesquiterpene alcohol that is present in various plants with a floral odor. It is... (Review)
Review
Nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol) is a naturally occurring sesquiterpene alcohol that is present in various plants with a floral odor. It is synthesized as an intermediate in the production of (3E)-4,8-dimethy-1,3,7-nonatriene (DMNT), a herbivore-induced volatile that protects plants from herbivore damage. Chemically, nerolidol exists in two geometric isomers, a trans and a cis form. The usage of nerolidol is widespread across different industries. It has been widely used in cosmetics (e.g., shampoos and perfumes) and in non-cosmetic products (e.g., detergents and cleansers). In fact, U.S. Food and Drug Administration (FDA) has also permitted the use of nerolidol as a food flavoring agent. The fact that nerolidol is a common ingredient in many products has attracted researchers to explore more medicinal properties of nerolidol that may exert beneficial effect on human health. Therefore, the aim of this review is to compile and consolidate the data on the various pharmacological and biological activities displayed by nerolidol. Furthermore, this review also includes pharmacokinetic and toxicological studies of nerolidol. In summary, the various pharmacological and biological activities demonstrated in this review highlight the prospects of nerolidol as a promising chemical or drug candidate in the field of agriculture and medicine.
Topics: Animals; Antioxidants; Cosmetics; Detergents; Flavoring Agents; Humans; Neoplasms; Oils, Volatile; Sesquiterpenes; United States; United States Food and Drug Administration
PubMed: 27136520
DOI: 10.3390/molecules21050529 -
Scientific Reports Jun 2022Roots and rhizomes can play an important role in nutrient cycling, however, few studies have investigated how their decomposition pattern is affected by defoliation and...
Roots and rhizomes can play an important role in nutrient cycling, however, few studies have investigated how their decomposition pattern is affected by defoliation and time of the year. This 2-year study evaluated root-rhizome composition and decomposition of a warm-season rhizomatous perennial legume [rhizoma peanut (RP; Arachis glabrata Benth.)] under continuous stocking or when defoliated by clipping every 56 days. A 168-days incubation trial was performed to determine disappearance of biomass and N and changes in acid detergent fiber (ADF), acid detergent insoluble N (ADIN), and C:N ratio. Additionally, three 56-days incubations were performed each year to evaluate the disappearance coefficient (B) and relative decay rate (k). There were no treatment differences in any response for the 168-days incubation. After 168 days, 21 and 60% of initial biomass and initial N remained, respectively. Relative decay rate for OM and N were 0.0088 and 0.0035 g g day, respectively. Carbon-to-N ratio decreased from 29 at day 0 to 17 at day 168. Concentration of ADIN increased from 6.9 to 19.3 g kg, plateauing at day 79. The B and k for remaining OM and N were greater in late than early season and could be explained by greater N concentration and lesser C:N ratio. Rapid decomposition, difference in C:N ratio from day 0 to 168, and the increase in ADIN concentration during incubation indicate large amounts of root-rhizome-soluble C at initiation of incubation. These data indicate that RP root-rhizome turnover is more responsive to season than defoliation frequency.
Topics: Arachis; Biomass; Carbon; Detergents; Fabaceae
PubMed: 35705653
DOI: 10.1038/s41598-022-14001-7 -
Biophysical Journal Apr 2023TolC is the trimeric outer membrane component of the efflux pump system in Escherichia coli that is responsible for antibiotic efflux from bacterial cells....
TolC is the trimeric outer membrane component of the efflux pump system in Escherichia coli that is responsible for antibiotic efflux from bacterial cells. Overexpression of efflux pumps has been reported to decrease susceptibility to antibiotics in a variety of bacterial pathogens. Reliable production of membrane proteins allows for the biophysical and structural characterization needed to better understand efflux and for the development of therapeutics. Preparation of recombinant protein for biochemical/structural studies often involves the production of proteins as inclusion body aggregates from which active proteins are recovered. Here, we find that the in vitro folding of TolC into its functional trimeric state from inclusion bodies is dependent on the headgroup composition of detergent micelles used. Nonionic detergent favors the formation of functional trimeric TolC, whereas zwitterionic detergents induce the formation of a non-native, oligomeric TolC fold. We also find that nonionic detergents with shorter alkyl lengths facilitate TolC folding. It remains to be seen whether the charges in lipid headgroups have similar effects on membrane insertion and folding in biological systems.
Topics: Membrane Transport Proteins; Detergents; Escherichia coli Proteins; Bacterial Outer Membrane Proteins; Escherichia coli; Anti-Bacterial Agents
PubMed: 36772796
DOI: 10.1016/j.bpj.2023.02.007