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Biochemical Society Transactions Jun 2023Membrane proteins are a highly relevant class of biological molecules and comprise ∼60% of current drug targets. Before being analyzed by structural, biochemical, and... (Review)
Review
Membrane proteins are a highly relevant class of biological molecules and comprise ∼60% of current drug targets. Before being analyzed by structural, biochemical, and biophysical methods, membrane proteins must first be extracted from cellular membranes - often using detergents. Detergent-extracted membrane proteins are amenable to analysis by structural, biochemical, and biophysical techniques. In certain cases, however, detergents can disturb native protein conformations and/or biological activity. This has led to the development of membrane mimetics, which stabilize membrane proteins in a native membrane-like environment that is water-soluble and detergent-free. This review provides an overview of recent developments in the membrane mimetic field, with a focus on nanodiscs, Saposin lipid nanoparticles (SapNPs), peptidiscs, and SMA lipid particles (SMALPs) - and highlights their utility for supporting biophysical, biochemical, and structural characterization of membrane proteins and complexes.
Topics: Membrane Proteins; Cell Membrane; Biomimetics; Lipid Bilayers; Detergents
PubMed: 37345653
DOI: 10.1042/BST20230164 -
Molecules (Basel, Switzerland) Nov 2022Bicelles are disk-shaped models of cellular membranes used to study lipid-protein interactions, as well as for structural and functional studies on transmembrane...
Bicelles are disk-shaped models of cellular membranes used to study lipid-protein interactions, as well as for structural and functional studies on transmembrane proteins. One challenge for the incorporation of transmembrane proteins in bicelles is the limited range of detergent and lipid combinations available for the successful reconstitution of proteins in model membranes. This is important, as the function and stability of transmembrane proteins are very closely linked to the detergents used for their purification and to the lipids that the proteins are embedded in. Here, we expand the toolkit of lipid and detergent combinations that allow the formation of stable bicelles. We use a combination of dynamic light scattering, small-angle X-ray scattering and cryogenic electron microscopy to perform a systematic sample characterization, thus providing a set of conditions under which bicelles can be successfully formed.
Topics: Lipid Bilayers; Surface-Active Agents; Detergents; Magnetic Resonance Spectroscopy; Micelles; Membrane Proteins; Pulmonary Surfactants
PubMed: 36364455
DOI: 10.3390/molecules27217628 -
Methods in Molecular Biology (Clifton,... 2022The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of...
The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments-are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher-order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton.This article describes application of rotary shadowing (or platinum replica ) EM (PREM) for visualization of the cytoskeleton . The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction (or mechanical "unroofing") of cells to expose their cytoskeleton , chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved and individual proteins can be identified by immunogold labeling. More importantly, PREM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high-resolution structural organization of the cytoskeleton in the same cell.
Topics: Actin Cytoskeleton; Actins; Cytoskeleton; Detergents; Microscopy, Electron; Microtubules; Platinum
PubMed: 34542847
DOI: 10.1007/978-1-0716-1661-1_2 -
Acta Crystallographica. Section F,... Dec 2014At the time when the first membrane-protein crystal structure was determined, crystallization of these molecules was widely perceived as extremely arduous. Today, that... (Review)
Review
At the time when the first membrane-protein crystal structure was determined, crystallization of these molecules was widely perceived as extremely arduous. Today, that perception has changed drastically, and the process is regarded as routine (or nearly so). On the occasion of the International Year of Crystallography 2014, this review presents a snapshot of the current state of the art, with an emphasis on the role of detergents in this process. A survey of membrane-protein crystal structures published since 2012 reveals that the direct crystallization of protein-detergent complexes remains the dominant methodology; in addition, lipidic mesophases have proven immensely useful, particularly in specific niches, and bicelles, while perhaps undervalued, have provided important contributions as well. Evolving trends include the addition of lipids to protein-detergent complexes and the gradual incorporation of new detergents into the standard repertoire. Stability has emerged as a critical parameter controlling how a membrane protein behaves in the presence of detergent, and efforts to enhance stability are discussed. Finally, although discovery-based screening approaches continue to dwarf mechanistic efforts to unravel crystallization, recent technical advances offer hope that future experiments might incorporate the rational manipulation of crystallization behaviors.
Topics: Crystallization; Crystallography; Detergents; Membrane Proteins; Protein Stability
PubMed: 25484203
DOI: 10.1107/S2053230X14025035 -
Brazilian Journal of Microbiology :... Jun 2023Detergent-compatible enzymes are the new trend followed by most in the detergent industry. Cellulases, lipases, proteases, and amylases are among the enzymes frequently...
Detergent-compatible enzymes are the new trend followed by most in the detergent industry. Cellulases, lipases, proteases, and amylases are among the enzymes frequently used in detergents. Detergent-compatible enzymes can be obtained from many organisms, but the stability, cheapness, and availability of microbial enzymes make them preferable in industrial areas. In the present study, soil samples contaminated with household waste were collected from different regions of Trabzon (Turkey) for amylase-, cellulase-, protease-, and lipase-producing bacteria. A total of 55 bacterial isolates differing in colony morphology were purified from the samples and 25 of the isolates gave positive results in enzyme screening. The enzyme screening experiments revealed that 10 isolates produced amylase, 9 produced lipase, 7 produced cellulase, and 6 produced protease. While 2 isolates showed both protease and lipase activity, for 2 different isolates cellulose and amylase activity were detected together. It was also observed that one isolate, C37PLCA, produced all four enzymes. The morphological, physiological, and biochemical analyses of the bacteria from which we obtained the enzymes were performed and species close to them were determined using 16S rRNA sequences. Based on the results obtained, our enzymes show tremendous promise for the detergent industry.
Topics: Peptide Hydrolases; Lipase; Detergents; Amylases; Cellulase; RNA, Ribosomal, 16S; Bacterial Proteins; Bacteria; Cellulases
PubMed: 36890351
DOI: 10.1007/s42770-023-00944-0 -
Current Opinion in Structural Biology Oct 2019Solving high-resolution structures of membrane proteins has been an important challenge for decades, still lagging far behind that of soluble proteins even with the... (Review)
Review
Solving high-resolution structures of membrane proteins has been an important challenge for decades, still lagging far behind that of soluble proteins even with the recent remarkable technological advances in X-ray crystallography and electron microscopy. Central to this challenge is the necessity to isolate and solubilize membrane proteins in a stable, natively folded and functional state, a process influenced by not only the proteins but also their surrounding chemical environment. This review highlights recent community efforts in the development and characterization of novel membrane agents and ligand tools to stabilize individual proteins and protein complexes, which together have accelerated progress in membrane protein structural biology.
Topics: Crystallization; Detergents; Membrane Proteins; Protein Stability; Solubility
PubMed: 31285102
DOI: 10.1016/j.sbi.2019.06.002 -
Methods (San Diego, Calif.) Sep 2018The last ∼25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural... (Review)
Review
The last ∼25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods.
Topics: Detergents; Mass Spectrometry; Membrane Proteins; Protein Conformation; Protein Folding
PubMed: 29510247
DOI: 10.1016/j.ymeth.2018.02.020 -
Acta Biochimica Et Biophysica Sinica Aug 2022Structure determination of membrane proteins has been a long-standing challenge to understand the molecular basis of life processes. Detergents are widely used to study... (Review)
Review
Structure determination of membrane proteins has been a long-standing challenge to understand the molecular basis of life processes. Detergents are widely used to study the structure and function of membrane proteins by various experimental methods, and the application of membrane mimetics is also a prevalent trend in the field of cryo-EM analysis. This review focuses on the widely-used detergents and corresponding properties and structures, and also discusses the growing interests in membrane mimetic systems used in cryo-EM studies, providing insights into the role of detergent alternatives in structure determination.
Topics: Cryoelectron Microscopy; Detergents; Membrane Proteins
PubMed: 35866608
DOI: 10.3724/abbs.2022088 -
Tropical Medicine & International... Apr 2023Arboviruses are emerging as a relevant threat to transfusion safety. Pathogen inactivation methods (PIMs) may reduce the risk of transmission through transfusion, as... (Meta-Analysis)
Meta-Analysis Review
OBJECTIVE
Arboviruses are emerging as a relevant threat to transfusion safety. Pathogen inactivation methods (PIMs) may reduce the risk of transmission through transfusion, as long as they meet minimum standards for effectiveness. This study aims to assess the log reduction of viral load achieved with different PIMs, according to the blood product they are used on and the arbovirus targeted.
METHODS
Systematic literature review and meta-analysis. Searches were conducted in MEDLINE and Embase. The study protocol was registered in PROSPERO CRD42022312061. We selected records reporting the log reduction of viral load achieved with the main PIMs (amotosalen + UVA light [INTERCEPT], riboflavin + UV light [Mirasol], methylene blue + visible light/UVC light [THERAFLEX], solvent detergent, amustaline [INTERCEPT] and PEN110 [Inactine]), applied to any blood product (plasma, platelets, red blood cells or whole blood) and for any arbovirus. The log reduction of viral loads was assessed by obtaining the mean log reduction factor (LRF). We compared and classified the LRF of different techniques using statistical methods.
RESULTS
We included 59 publications reporting LRF results in 17 arboviruses. For 13 arboviruses, including Chikungunya virus, Dengue virus, West Nile virus and Zika virus, at least one of the methods achieves adequate or optimal log reduction of viral load-mean LRF ≥4. The LRF achieved with riboflavin + UV light is inferior to the rest of the techniques, both overall and specifically for plasma, platelets preserved in platelet additive solution (PAS)/plasma, and red blood cells/whole blood. The LRF achieved using Mirasol is also lower for inactivating Chikungunya virus, Dengue virus and Zika virus. For West Nile virus, we found no significant differences. In plasma, the method that achieves the highest LRF is solvent/detergent; in platelets, THERAFLEX and INTERCEPT; and in red blood cells/whole blood, PEN110 (Inactine).
CONCLUSION
Not all PIMs achieve the same LRF, nor is this equivalent between the different arboviruses or blood products. Overall, the LRFs achieved using riboflavin + UV light (Mirasol) are inferior to those achieved with the rest of the PIMs. Regarding the others, LRFs vary by arbovirus and blood product. In light of the threat of different arboviruses, blood establishments should have already validated PIMs and be logistically prepared to implement these techniques quickly.
Topics: Humans; Arboviruses; Detergents; Polyamines; Zika Virus; Riboflavin; Zika Virus Infection
PubMed: 36806816
DOI: 10.1111/tmi.13863 -
Science China. Life Sciences Jan 2015Membrane proteins are involved in various critical biological processes, and studying membrane proteins represents a major challenge in protein biochemistry. As shown by... (Review)
Review
Membrane proteins are involved in various critical biological processes, and studying membrane proteins represents a major challenge in protein biochemistry. As shown by both structural and functional studies, the membrane environment plays an essential role for membrane proteins. In vitro studies are reliant on the successful reconstitution of membrane proteins. This review describes the interaction between detergents and lipids that aids the understanding of the reconstitution processes. Then the techniques of detergent removal and a few useful techniques to refine the formed proteoliposomes are reviewed. Finally the applications of reconstitution techniques to study membrane proteins involved in Ca(2+) signaling are summarized.
Topics: Detergents; In Vitro Techniques; Membrane Proteins; Microscopy, Electron; Structure-Activity Relationship
PubMed: 25576454
DOI: 10.1007/s11427-014-4769-0