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Chemistry (Weinheim An Der Bergstrasse,... Dec 2022It is a pressing need, but still challenging to explore the structure and function of membrane proteins (MPs). One of the main obstacles is the limited availability of...
It is a pressing need, but still challenging to explore the structure and function of membrane proteins (MPs). One of the main obstacles is the limited availability of matched detergents for the handling of specific MPs. We describe herein the design of new detergents by incorporation of a transition linker between the hydrophilic head and the hydrophobic tail. This design allows a gradual change of hydrophobicity between the outside and inside of micelles, in contrast to the abrupt switch in conventional detergents. Notably, many of these detergents assembled into micelles in while retaining low critical micelle concentrations. Meanwhile, thermal stabilizing evaluation identified superior detergents for representative MPs, including G protein-coupled receptors and a transporter protein. Among them, further improved the NMR study of MPs. We anticipate these that results will encourage future detergent expansion through new remodeling on the traditional detergent scaffold.
Topics: Detergents; Membrane Proteins; Micelles; Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Spectroscopy
PubMed: 36053145
DOI: 10.1002/chem.202202242 -
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 -
Archives of Biochemistry and Biophysics Dec 2014Amphipols (APols) are a family of amphipathic polymers designed to keep transmembrane proteins (TMPs) soluble in aqueous solutions in the absence of detergent. APols... (Review)
Review
Amphipols (APols) are a family of amphipathic polymers designed to keep transmembrane proteins (TMPs) soluble in aqueous solutions in the absence of detergent. APols have proven remarkably efficient at (i) stabilizing TMPs, as compared to detergent solutions, and (ii) folding them from a denatured state to a native, functional one. The underlying physical-chemical mechanisms are discussed.
Topics: Detergents; Membrane Proteins; Protein Denaturation; Protein Folding; Protein Stability; Solubility
PubMed: 25449655
DOI: 10.1016/j.abb.2014.10.013 -
Methods in Molecular Biology (Clifton,... 2016The hypothesis that the Golgi apparatus is capable of sorting proteins and sending them to the plasma membrane through "lipid rafts," membrane lipid domains highly...
The hypothesis that the Golgi apparatus is capable of sorting proteins and sending them to the plasma membrane through "lipid rafts," membrane lipid domains highly enriched in glycosphingolipids, sphingomyelin, ceramide, and cholesterol, was formulated by van Meer and Simons in 1988 and came to a turning point when it was suggested that lipid rafts could be isolated thanks to their resistance to solubilization by some detergents, namely Triton X-100. An incredible number of papers have described the composition and properties of detergent-resistant membrane fractions. However, the use of this method has also raised the fiercest criticisms. In this chapter, we would like to discuss the most relevant methodological aspects related to the preparation of detergent-resistant membrane fractions, and to discuss the importance of discriminating between what is present on a cell membrane and what we can prepare from cell membranes in a laboratory tube.
Topics: Cell Fractionation; Cell Membrane; Detergents; Membrane Microdomains; Membrane Proteins; Temperature
PubMed: 26552679
DOI: 10.1007/978-1-4939-3170-5_10 -
Tissue Engineering. Part C, Methods Jun 2022Due to the abundance of bioactive components, surficial decoration with cell-derived extracellular matrix (ECM) is a promising strategy to improve the biological...
Due to the abundance of bioactive components, surficial decoration with cell-derived extracellular matrix (ECM) is a promising strategy to improve the biological functionality of the tissue engineering scaffolds. However, decellularization is necessary to remove antigenic components in the ECM that may trigger adverse immune response. Freeze-thaw (FT) cycles and treatment with Triton X-100/ammonium hydroxide (TN) are two commonly used decellularization methods for ECM, but their effects on both growth factor retention and antigen removal are still controversial. The objectives of this study are to compare the preservation of ECM texture and beneficial ingredients and the removal of cellular antigens by these two methods. First, the constructs combined bone marrow mesenchymal stem cell-derived ECM and poly(lactic-co-glycolic acid) (PLGA) membrane are prepared and decellularized using FT and TN treatments. Moreover, the effects of decellularization on the ultrastructure and the composition of ECM-decorated PLGA membrane are compared by scanning electron microscope observation and protein quantification. Furthermore, the ECM deposited on PLGA is stripped off and then implanted subcutaneously in rats, and the host macrophage and local lymphocyte responses were investigated. Finally, ECM-decorated porous PLGA scaffolds are implanted into rat calvarial defects, and the new bone formation is evaluated. Our results showed that both methods effectively removed DNA. TN treatment partially retained collagen, glycosaminoglycan, bone morphogenetic protein-2, and vascular endothelial growth factor, and better preserved structural integrity than FT treatment. ECM implants decellularized by both methods induced a mild host response after subcutaneous implantation. Although the total content of residual DNA in the two ECMs digested by the DNA enzyme seemed to be similar and very low, the interfaces between implanted materials and natural tissues in the TN group recruited lower numbers of CD68 macrophages, CD68CD86 (M1) macrophages, and CD4 T lymphocytes than that in FT group, implying that there exist other ECM antigens to influence immune response besides DNA. Furthermore, ECM-decorated scaffolds decellularized by TN treatment induced greater bone formation than that of bare scaffolds , demonstrating the effective retention of ECM bioactive components after decellularization. This study showed that TN treatment was a more effective and safer decellularization method than FT cycles. Impact statement Decellularization is a prerequisite for extracellular matrix (ECM) application, but there is still no standard for its selection. This study demonstrated that detergent treatment was more effective than freeze-thaw (FT) cycles in removing ECM antigens besides DNA, and the prepared ECM elicited a milder allogenic immune response, which ensured the safety of ECM. Moreover, detergent better preserved the ECM integrity than FT cycles, and effectively retained growth factors, and the decellularized ECM-decorated scaffolds significantly promoted bone repair, which ensured the effectiveness of ECM. This study provides the theoretical and experimental bases for the decellularization strategy of ECM-modified tissue engineering scaffolds.
Topics: Animals; DNA; Detergents; Extracellular Matrix; Rats; Tissue Engineering; Tissue Scaffolds; Vascular Endothelial Growth Factor A
PubMed: 35596569
DOI: 10.1089/ten.TEC.2022.0025 -
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 -
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) May 2021Solubilization of carbon nanotubes (CNTs) is a fundamental technique for the use of CNTs and their conjugates as nanodevices and nanobiodevices. In this work, we...
Solubilization of carbon nanotubes (CNTs) is a fundamental technique for the use of CNTs and their conjugates as nanodevices and nanobiodevices. In this work, we demonstrate the preparation of CNT suspensions with "green" detergents made from coconuts and bamboo as fundamental research in CNT nanotechnology. Single-walled CNTs (SWNTs) with a few carboxylic acid groups (3-5%) and pristine multi-walled CNTs (MWNTs) were mixed in each detergent solution and sonicated with a bath-type sonicator. The prepared suspensions were characterized using absorbance spectroscopy, scanning electron microscopy, and Raman spectroscopy. Among the eight combinations of CNTs and detergents (two types of CNTs and four detergents, including sodium dodecyl sulfate (SDS) as the standard), SWNTs/MWNTs were well dispersed in all combinations except the combination of the MWNTs and the bamboo detergent. The stability of the suspensions prepared with coconut detergents was better than that prepared with SDS. Because the efficiency of the bamboo detergents against the MWNTs differed significantly from that against the SWNTs, the natural detergent might be useful for separating CNTs. Our results revealed that the use of the "green" detergents had the advantage of dispersing CNTs as well as SDS.
Topics: Centrifugation; Detergents; Nanotubes, Carbon; Suspensions
PubMed: 34068851
DOI: 10.3390/molecules26102908 -
Chembiochem : a European Journal of... Apr 2022Integral membrane proteins pose considerable challenges to high resolution structural analysis. Maintaining membrane proteins in their native state during protein...
Integral membrane proteins pose considerable challenges to high resolution structural analysis. Maintaining membrane proteins in their native state during protein isolation is essential for structural study of these bio-macromolecules. Detergents are the most commonly used amphiphilic compounds for stabilizing membrane proteins in solution outside a lipid bilayer. We previously introduced a glyco-diosgenin (GDN) detergent that was shown to be highly effective at stabilizing a wide range of membrane proteins. This steroidal detergent has additionally gained attention due to its compatibility with membrane protein structure study via cryo-EM. However, synthetic inconvenience limits widespread use of GDN in membrane protein study. To improve its synthetic accessibility and to further enhance detergent efficacy for protein stabilization, we designed a new class of glyco-steroid-based detergents using three steroid units: cholestanol, cholesterol and diosgenin. These new detergents were efficiently prepared and showed marked efficacy for protein stabilization in evaluation with a few model membrane proteins including two G protein-coupled receptors. Some new agents were not only superior to a gold standard detergent, DDM (n-dodecyl-β-d-maltoside), but were also more effective than the original GDN at preserving protein integrity long term. These agents represent valuable alternatives to GDN, and are likely to facilitate structural determination of challenging membrane proteins.
Topics: Detergents; Hydrophobic and Hydrophilic Interactions; Membrane Proteins; Protein Stability; Steroids
PubMed: 35129249
DOI: 10.1002/cbic.202200027