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Journal of Chemical Information and... Nov 2023Membrane proteins are difficult to isolate and purify due to their dependence on the surrounding lipid membrane for structural stability. Detergents are often used to...
Membrane proteins are difficult to isolate and purify due to their dependence on the surrounding lipid membrane for structural stability. Detergents are often used to solubilize these proteins, with this approach requiring a careful balance between protein solubilization and denaturation. Determining which detergent is most appropriate for a given protein has largely been done empirically through screening, which requires large amounts of membrane protein and associated resources. Here, we describe an alternative to conventional detergent screening using a computational modeling approach to identify the most likely candidate detergents for solubilizing a protein of interest. We demonstrate our approach using ghrelin -acyltransferase (GOAT), a member of the membrane-bound -acyltransferase family of integral membrane enzymes that has not been solubilized or purified in active form. A computationally derived GOAT structural model provides the only structural information required for this approach. Using computational analysis of detergent ability to penetrate phospholipid bilayers and stabilize the GOAT structure, a panel of common detergents were rank-ordered for their proposed ability to solubilize GOAT. The simulations were performed at all-atom resolution for a combined simulation time of 24 μs. Independently, we biologically screened these detergents for their solubilization of fluorescently tagged GOAT constructs. We found computational prediction of protein structural stabilization was the better predictor of detergent solubilization ability, but neither approach was effective for predicting detergents that would support GOAT enzymatic function. The current rapid expansion of membrane protein computational models lacking experimental structural information and our computational detergent screening approach can greatly improve the efficiency of membrane protein detergent solubilization, supporting downstream functional and structural studies.
Topics: Animals; Detergents; Membrane Proteins; Phospholipids; Acyltransferases; Goats; Solubility
PubMed: 37939203
DOI: 10.1021/acs.jcim.3c00917 -
Langmuir : the ACS Journal of Surfaces... Mar 2019We report herein the design and synthesis of a novel series of alkyl glycoside detergents consisting of a nonionic polar headgroup that comprises two glucose moieties in...
We report herein the design and synthesis of a novel series of alkyl glycoside detergents consisting of a nonionic polar headgroup that comprises two glucose moieties in a branched arrangement (DG), onto which octane-, decane-, and dodecanethiols were grafted leading to ODG, DDG, and DDDG detergents, respectively. Micellization in aqueous solution was studied by isothermal titration calorimetry, H NMR spectroscopy, and surface tensiometry. Critical micellar concentration values were found to decrease by a factor of ∼10 for each pair of methylene groups added to the alkyl chain, ranging from ∼0.05 to 9 mM for DDDG and ODG, respectively. Dynamic light scattering and analytical ultracentrifugation sedimentation velocity experiments were used to investigate the size and composition of the micellar aggregates, showing that the aggregation number significantly increased from ∼40 for ODG to ∼80 for DDDG. All new compounds were able to solubilize membrane proteins (MPs) from bacterial membranes, insect cells, as well as the Madin-Darby canine kidney cells. In particular, native human adenosine receptor (A2AR) and bacterial transporter (BmrA) were solubilized efficiently. Striking thermostability improvements of +13 and +8 °C were observed when ODG and DDG were, respectively, applied to wild-type and full-length A2AR. Taken together, this novel detergent series shows promising detergent potency for solubilization and stabilization of membrane proteins (MPs) and thus makes a valuable addition to the chemical toolbox available for extracting and handling these important but challenging MP targets.
Topics: Detergents; Glucose; Hydrogenation; Membrane Proteins; Particle Size; Protein Stability; Surface Properties
PubMed: 30767533
DOI: 10.1021/acs.langmuir.8b02842 -
Journal of Animal Science Jan 2023This study evaluated the effects of physically effective neutral detergent fiber (peNDF) and undigested neutral detergent fiber (uNDF) on eating behavior, ruminal...
Effect of physically effective neutral detergent fiber and undigested neutral detergent fiber on eating behavior, ruminal fermentation and motility, barrier function, blood metabolites, and total tract digestibility in finishing cattle.
This study evaluated the effects of physically effective neutral detergent fiber (peNDF) and undigested neutral detergent fiber (uNDF) on eating behavior, ruminal fermentation and motility, barrier function, blood metabolites, and total tract nutrient digestibility for finishing cattle. Six Simmental heifers (668 ± 28.4 kg BW) were used in a replicated 3 × 3 Latin square (21 d periods) balanced for carry-over effects. Treatments included a control (CON; Table 1) with no forage peNDF and minimal uNDF (peNDF: 0.0%, and uNDF: 4.88 ± 0.01; 95.15% barley grain, 4.51% vitamin and mineral supplement, and 0.34% urea on a DM basis). Pelleted wheat straw (PELL) was included at 10% of dietary DM by replacing barley grain to provide added uNDF but no forage peNDF (peNDF: 0.00%, and uNDF: 6.78 ± 0.02%). Finally, chopped wheat straw (STR) was included as a replacement for pelleted wheat straw to provide forage peNDF and uNDF (peNDF: 1.74 ± 0.06%, and uNDF: 6.86 ± 0.03%). Dry matter intake was not affected (P = 0.93) by treatments. Cattle fed CON spent less time ruminating (P = 0.010) and had less meals/d (P = 0.035) when compared with cattle fed STR, with those fed PELL being intermediate but not different from other treatments. Cattle fed CON had lesser ruminal pH (P = 0.020), and a greater duration that pH was < 5.5 (P = 0.020) as compared to cattle fed STR, with those fed PELL being intermediate but not different. Cattle fed CON and PELL had greater total short-chain fatty acid concentration (P = 0.003) and molar proportion of propionate (P < 0.001) when compared with cattle fed STR. Cattle fed STR had greater (P = 0.010) total ruminal pool size when compared with cattle fed CON and PELL. Cattle fed CON had greater (P = 0.043) duration between ruminal contractions when compared with cattle fed STR, with those fed PELL being intermediate. Cattle fed CON had greater serum amyloid A (P = 0.003) and haptoglobin (P < 0.001) concentration when compared with the other treatments. Cattle fed CON had greater dry matter (P < 0.001) digestibility when compared with the other treatments. In conclusion, inclusion of PELL and STR impacted eating behavior, but only STR affected ruminal fermentation, ruminal motility, systemic inflammation, and total tract nutrient digestibility in finishing cattle. These results are interpreted to suggest that the combination of peNDF and uNDF may be better than uNDF alone to adequately capture biological effects of NDF in high-concentrate diets.
Topics: Animals; Cattle; Female; Animal Feed; Detergents; Diet; Dietary Fiber; Digestion; Feeding Behavior; Fermentation; Hydrogen-Ion Concentration; Rumen; Triticum
PubMed: 36638278
DOI: 10.1093/jas/skad023 -
Cells Jul 2023The application of decellularized scaffolds for artificial tissue reconstruction has been an approach with great therapeutic potential in regenerative medicine....
The application of decellularized scaffolds for artificial tissue reconstruction has been an approach with great therapeutic potential in regenerative medicine. Recently, biomimetic ovarian tissue reconstruction was proposed to reestablish ovarian endocrine functions. Despite many decellularization methods proposed, there is no established protocol for whole ovaries by detergent perfusion that is able to preserve tissue macro and microstructure with higher efficiency. This generated biomaterial may have the potential to be applied for other purposes beyond reproduction and be translated to other areas in the tissue engineering field. Therefore, this study aimed to establish and standardize a protocol for porcine ovaries' decellularization based on detergent perfusion and ultrasonication to obtain functional whole-ovary scaffolds. For that, porcine ovaries ( = 5) were perfused with detergents (0.5% SDS and 1% Triton X-100) and submitted to an ultrasonication bath to produce acellular scaffolds. The decellularization efficiency was evaluated by DAPI staining and total genomic DNA quantification. ECM morphological evaluation was performed by histological, immunohistochemistry, and ultrastructural analyses. ECM physico-chemical composition was evaluated using FTIR and Raman spectroscopy. A cytocompatibility and cell adhesion assay using murine fibroblasts was performed. Results showed that the proposed method was able to remove cellular components efficiently. There was no significant ECM component loss in relation to native tissue, and the scaffolds were cytocompatible and allowed cell attachment. In conclusion, the proposed decellularization protocol produced whole-ovaries scaffolds with preserved ECM composition and great potential for application in tissue engineering.
Topics: Female; Swine; Mice; Animals; Ovary; Tissue Scaffolds; Detergents; Extracellular Matrix; Perfusion
PubMed: 37508528
DOI: 10.3390/cells12141864 -
Biochemical Society Transactions Apr 2020A growing number of integral membrane proteins have been shown to tune their activity by selectively interacting with specific lipids. The ability to regulate biological... (Review)
Review
A growing number of integral membrane proteins have been shown to tune their activity by selectively interacting with specific lipids. The ability to regulate biological functions via lipid interactions extends to the diverse group of proteins that associate only peripherally with the lipid bilayer. However, the structural basis of these interactions remains challenging to study due to their transient and promiscuous nature. Recently, native mass spectrometry has come into focus as a new tool to investigate lipid interactions in membrane proteins. Here, we outline how the native MS strategies developed for integral membrane proteins can be applied to generate insights into the structure and function of peripheral membrane proteins. Specifically, native MS studies of proteins in complex with detergent-solubilized lipids, bound to lipid nanodiscs, and released from native-like lipid vesicles all shed new light on the role of lipid interactions. The unique ability of native MS to capture and interrogate protein-protein, protein-ligand, and protein-lipid interactions opens exciting new avenues for the study of peripheral membrane protein biology.
Topics: Antigens, CD1; Antimicrobial Cationic Peptides; Binding Sites; Detergents; Glycosphingolipids; Humans; Lipid Bilayers; Lipids; Mass Spectrometry; Membrane Proteins; Peptides; Protein Interaction Mapping; Spectrometry, Mass, Electrospray Ionization; Ubiquinone
PubMed: 32129823
DOI: 10.1042/BST20190787 -
Analytical Sciences : the International... Aug 2022Detergent removal in glycolipid after sample preparation, such as enzymatic reaction or isolation of detergent-resistant membrane microdomain, is indispensable for...
Detergent removal in glycolipid after sample preparation, such as enzymatic reaction or isolation of detergent-resistant membrane microdomain, is indispensable for further structural characterization. We previously established the rapid and effective method of detergent removal in glycolipid samples from glass test tube using 1,2-dichloroethane (DCE) washing. However, the use of DCE has several drawbacks, such as environmental risks, harmful effects (potentially carcinogenic), and high vaporability and flammability. To solve the issue, we used ionic liquids to remove detergents from glycolipid samples, and found 1-butyl-3-methylimidazolium iodide was a suitable alternative for DCE.
Topics: Detergents; Glycolipids; Iodides; Ionic Liquids
PubMed: 35718829
DOI: 10.1007/s44211-022-00139-x -
Methods in Molecular Biology (Clifton,... 2022The development of styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers provides an alternative to traditional detergent extraction of integral...
The development of styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers provides an alternative to traditional detergent extraction of integral membrane proteins. By inserting into the membrane, these polymers can extract membrane proteins along with lipids in the form of native nanodiscs made by poly(styrene co-maleic anhydride) derivatives. Unlike detergent solubilization, where membrane proteins may lose annular lipids necessary for proper folding and stability, native nanodiscs allow for proteins to reside in the natural lipid environment. In addition, polymer-based nanodiscs can be purified using common chromatography methods similar to protocols established with detergent solubilization purification. Here we describe the solubilization screening and purification of an integral membrane protein using several commercial copolymers.
Topics: Detergents; Lipid Bilayers; Lipids; Membrane Proteins; Polymers; Polystyrenes; Styrenes
PubMed: 35773593
DOI: 10.1007/978-1-0716-2368-8_20 -
Science China. Life Sciences Aug 2022Silage maize (Zea mays L.) is one of the most important forages in the world, and its yield and quality properties are critical parameters for livestock production and...
Silage maize (Zea mays L.) is one of the most important forages in the world, and its yield and quality properties are critical parameters for livestock production and assessment of forage values. However, relationships between its yield and quality properties and the controlling factors are not well documented. In this study, we collected 5,663 observations from 196 publications across the country to identify the relationships between yield and quality properties of silage maize and to assess the impact of management practices and climatic factors on its yield and quality in China. The average dry matter yield of silage maize was (19.98±6.93) Mg ha, and the average value of crude protein, ether extract, crude ash, crude fiber, acid detergent fiber, neutral detergent fiber, nitrogen-free extract, and relative feed value was 7.86%±1.71%, 2.53%±1.01%, 5.05%±1.66%, 23.97%±6.34%, 27.62%±7.12%, 51.60%±9.85%, 59.68%±7.72%, and 131.17±31.49, respectively. In general, its nutritive value decreased as its yield increased. Increasing planting density could increase the yield but inhibit the nutritive values, while increasing fertilization could benefit the nutritive values. Geographically, the yield increased and the nutritive value decreased from warm (south) to cold (north) regions. The length of growth duration was a major controlling factor for the patterns of these properties. Our findings provide insights for police-makers to make strategy for achieving high yield and good quality of silage maize and help local people to implement better management practices.
Topics: Detergents; Dietary Fiber; Humans; Nutritive Value; Silage; Zea mays
PubMed: 35122623
DOI: 10.1007/s11427-020-2023-3 -
CJEM Jan 2021
Topics: Child; Detergents; Humans; Vomiting
PubMed: 33683601
DOI: 10.1007/s43678-020-00032-4 -
Microbial Biotechnology May 2022A form of lateral membrane compartmentalization in bacteria is represented by functional membrane microdomains (FMMs). FMMs are important for various cellular processes...
A form of lateral membrane compartmentalization in bacteria is represented by functional membrane microdomains (FMMs). FMMs are important for various cellular processes and offer application possibilities in microbial biotechnology. We designed a lipidomics method to directly measure relative abundances of lipids in detergent-resistant and detergent-sensitive membrane fractions of the model bacterium Bacillus subtilis 168 and the biotechnologically attractive miniBacillus PG10 strain. Our study supports previous work suggesting that cardiolipin and prenol lipids are enriched in FMMs of B. subtilis. Additionally, structural analysis of acyl chains of major phospholipids indicated that FMMs display increased order and thickness compared with the surrounding bilayer. Despite the 36% genome reduction, membrane and FMM integrity are largely preserved in miniBacillus PG10, as supported by analysis of membrane fluidity, flotillin distribution and gene expression data. The novel insights in FMM architecture reported here will contribute to further explore the biological significance of FMMs and the means by which FMMs can be exploited as heterologous production platforms. Moreover, our lipidomics method enables comparative FMM lipid profiling between different bacteria.
Topics: Bacillus subtilis; Detergents; Membrane Fluidity; Membrane Microdomains
PubMed: 34856064
DOI: 10.1111/1751-7915.13978