scholarly journals Optimization of Membrane Protein TmrA Purification Procedure Guided by Analytical Ultracentrifugation

Membranes ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 780
Author(s):  
Dongdong Li ◽  
Wendan Chu ◽  
Xinlei Sheng ◽  
Wenqi Li
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Analytical Ultracentrifugation ◽  
Cost Effective ◽  
Effective Strategy ◽  
High Concentration ◽  
Aggregation State ◽  
Cellular Processes ◽  
Membrane Protein Stability ◽  
Triton X

Membrane proteins are involved in various cellular processes. However, purification of membrane proteins has long been a challenging task, as membrane protein stability in detergent is the bottleneck for purification and subsequent analyses. Therefore, the optimization of detergent conditions is critical for the preparation of membrane proteins. Here, we utilize analytical ultracentrifugation (AUC) to examine the effects of different detergents (OG, Triton X-100, DDM), detergent concentrations, and detergent supplementation on the behavior of membrane protein TmrA. Our results suggest that DDM is more suitable for the purification of TmrA compared with OG and TritonX-100; a high concentration of DDM yields a more homogeneous protein aggregation state; supplementing TmrA purified with a low DDM concentration with DDM maintains the protein homogeneity and aggregation state, and may serve as a practical and cost-effective strategy for membrane protein purification.

scholarly journals Membrane Protein Stabilization Strategies for Structural and Functional Studies

Membranes ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 155
Author(s):  
Ekaitz Errasti-Murugarren ◽  
Paola Bartoccioni ◽  
Manuel Palacín
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Membrane ◽  
Protein Stabilization ◽  
New Drugs ◽  
Cell Physiology ◽  
Protein Preparation ◽  
Functional Studies ◽  
Membrane Protein Stability ◽  
Druggable Targets

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

scholarly journals The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Michael Luke Carlson ◽  
John William Young ◽  
Zhiyu Zhao ◽  
Lucien Fabre ◽  
Daniel Jun ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Cost Effective ◽  
Water Soluble ◽  
Protein Assemblies ◽  
Simple Method ◽  
Helical Peptide ◽  
Biological Studies ◽  
Multiple Copies ◽  
Exposed Part

Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this ‘one size fits all’ method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during ‘on-column’, ‘in-gel’, and ‘on-bead’ reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.

scholarly journals A comparative study of branched and linear mannitol-based amphiphiles on membrane protein stability

The Analyst ◽  
2018 ◽  
Vol 143 (23) ◽  
pp. 5702-5710 ◽  
Author(s):  
Hazrat Hussain ◽  
Tyler Helton ◽  
Yang Du ◽  
Jonas S. Mortensen ◽  
Parameswaran Hariharan ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Comparative Study ◽  
Protein Stability ◽  
Alkyl Chain ◽  
Alkyl Chains ◽  
Membrane Protein Stability ◽  
The Comparative Study

The comparative study on linear vs. branched alkyl-chain amphiphiles indicates a favorable role for branched alkyl-chains in stabilizing membrane proteins.

scholarly journals Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins.

1985 ◽  
Vol 33 (8) ◽  
pp. 813-820 ◽  
Author(s):  
K L Goldenthal ◽  
K Hedman ◽  
J W Chen ◽  
J T August ◽  
M C Willingham
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Membrane Glycoproteins ◽  
Animal Cells ◽  
Cytoplasmic Proteins ◽  
Membrane Antigens ◽  
Endocytic Vesicles ◽  
Triton X ◽  
Nonionic Detergents

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.

Nanodisc characterization by analytical ultracentrifugation

2017 ◽  
Vol 6 (1) ◽  
pp. 3-14 ◽  
Author(s):  
Sayaka Inagaki ◽  
Rodolfo Ghirlando
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Analytical Ultracentrifugation ◽  
Size Composition ◽  
Functional Studies ◽  
Lipid Environment ◽  
The Many ◽  
Sample Homogeneity ◽  
Simple Fashion

AbstractDue to their unique properties, tunable size, and ability to provide a near native lipid environment, nanodiscs have found widespread use for the structural and functional studies of reconstituted membrane proteins. They have also been developed, albeit in a few applications, for therapeutic and biomedical use. For these studies and applications, it is essential to characterize the nanodisc preparations in terms of their monodispersity, size, and composition, as these can influence the properties of the membrane protein of interest. Of the many biophysical methods utilized for the study and characterization of nanodiscs, we show that analytical ultracentrifugation is able to report on sample homogeneity, shape, size, composition, and membrane protein stoichiometry or oligomerization state in a direct and simple fashion. The method is truly versatile and does not require nanodisc modification or disassembly.

Analytical Ultracentrifugation of Proteins in Solutions of Triton X-100 Using Absorption Optics

1985 ◽  
Vol 40 (11-12) ◽  
pp. 908-911 ◽  
Author(s):  
Dieter Schubert ◽  
Karin Boss
Keyword(s):  
Molecular Weight ◽  
Membrane Proteins ◽  
Path Length ◽  
Analytical Ultracentrifugation ◽  
Scanning System ◽  
Reference Cell ◽  
Solvent Density ◽  
Nonionic Detergent ◽  
D 12 ◽  
Triton X

Abstract Analytical ultracentrifugation studies on proteins in solutions of the nonionic detergent Triton X-100, by means of a UV scanning system, can be conveniently performed by the following procedure: (1) Scanning of the cells is done at a wavelength of 246 nm. (2) The sedimentation of the deter­ gent is prevented by appropriate adjustment of the solvent density. (3) The reference cell is filled with water. Reliable molecular weight determinations can be performed at detergent concentrations up to 0.4% (w/w) in cells of path-length d = 12 mm and at correspondingly higher concentrations for d - 3 mm. Approx. 10 μg of protein are sufficient for a determination. The method should be useful for studies of membrane proteins.

Lipid mediators in membrane rafts are important determinants of human health and disease

2007 ◽  
Vol 32 (3) ◽  
pp. 341-350 ◽  
Author(s):  
David W.L. Ma
Keyword(s):  
Membrane Proteins ◽  
Human Health ◽  
Spatial Organization ◽  
Saturated Fatty Acids ◽  
Dietary Lipids ◽  
Membrane Rafts ◽  
High Concentration ◽  
Metabolic Disturbances ◽  
Cellular Processes ◽  
And Function

The new field of membrane rafts has provided fresh insight and a novel framework in which to understand the interaction, relation, and organization of lipids and proteins within cell membranes. This review will examine our current understanding of membrane rafts and their role in human health. In addition, the effect of various lipids, including dietary lipids, on membrane raft structure and function will be discussed. Membrane rafts are found in all cells and are characterized by their high concentration of cholesterol, sphingolipids, and saturated fatty acids. These lipids impart lateral segregation of membrane proteins, thus facilitating the spatial organization and regulation of membrane proteins involved in many cellular processes, such as cell proliferation, apoptosis, and cell signaling. Therefore, membrane rafts are shedding new light on the origins of metabolic disturbances and diseases such as cancer, insulin resistance, inflammation, cardiovascular disease, and Alzheimer’s disease, which will be further discussed in this review.

scholarly journals Detection and characterization of monoclonal antibodies to platelet membrane proteins.

1981 ◽  
Vol 90 (1) ◽  
pp. 249-253 ◽  
Author(s):  
P J Newman ◽  
R A Kahn ◽  
A Hines
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Solid Phase ◽  
Platelet Membrane ◽  
Binding Assays ◽  
Platelet Surface ◽  
Before And After ◽  
Triton X

We have devised a solid-phase radioimmunoassay for the detection and characterization of monoclonal antibodies directed against platelet surface antigens. Platelet membrane proteins, solubilized with 0.1% Triton X-100, were covalently coupled to cyanogen bromide (CNBr)-activated filter paper disks that were than used as the support in antibody binding assays. SDS PAGE of solubilized membrane proteins taken immediately before and after incubation with activated disks indicated that representative amounts of each membrane protein were bound to the disks. Either monoclonal or heterologous anti-platelet antibody could be detected on disks that had been prepared using as little as 50 micrograms of membrane protein per 100 disks. For the detection of antibody, disks were incubated with test sera for 2 h, washed, and incubated with 125I-labeled anti-immunoglobulin G, and the amount of bound radioactivity was determined. The sensitivity of the disk assay in detecting monoclonal antibodies was far greater than that of a corresponding radioimmunoassay that used whole platelets as the solid phase. By linking other proteins such as fibrinogen or anti-mouse subclass specific antisera to CNBr-activated disks, the method was adapted for antibody characterization. The sensitivity and ease with which the assay can be performed make this technique most suitable for screening and characterizing monoclonal antibodies.

scholarly journals Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens

1975 ◽  
Vol 152 (3) ◽  
pp. 523-528 ◽  
Author(s):  
H G Hansma ◽  
C Kung
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Electrophoretic Pattern ◽  
Surface Protein ◽  
Major Band ◽  
Electrophoretic Mobilities ◽  
Immobilization Antigen ◽  
Triton X

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

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