scholarly journals Author Correction: Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Anna J. Higgins ◽  
Alex J. Flynn ◽  
Anaïs Marconnet ◽  
Laura J. Musgrove ◽  
Vincent L. G. Postis ◽  
...  
2021 ◽  
Author(s):  
Anna Higgins ◽  
Alex Flynn ◽  
Anaïs Marconnet ◽  
Laura Musgrove ◽  
Vincent Postis ◽  
...  

Abstract Membrane proteins are essential for cellular growth and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates significant challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a significant advantage. Here we use a novel type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful detergent-free approach for the study of membrane proteins allowing native extraction and high-resolution structure determination by cryoEM.


2018 ◽  
Vol 54 (97) ◽  
pp. 13702-13705 ◽  
Author(s):  
Nils Hellwig ◽  
Oliver Peetz ◽  
Zainab Ahdash ◽  
Igor Tascón ◽  
Paula J. Booth ◽  
...  

Other than more widely used methods, the use of styrene maleic acid copolymers allows the direct extraction of membrane proteins from the lipid bilayer into SMALPs keeping it in its native lipid surrounding.


2021 ◽  
Author(s):  
Thirupathi Ravula ◽  
Ayyalusamy Ramamoorthy

ABSTRACTA family of non-ionic amphiphilic polymers synthesized by hydrophobic functionalization of fructo-oligosaccharides/inulin is reported. We show the stability of lipid-nanodiscs formed by these polymers against pH and divalent metal ions, and their magnetic-alignment properties. The reported results also demonstrate that the non-ionic polymers extract membrane proteins with unprecedented efficiency.


2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Anna J. Higgins ◽  
Alex J. Flynn ◽  
Anaïs Marconnet ◽  
Laura J. Musgrove ◽  
Vincent L. G. Postis ◽  
...  

AbstractMembrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.


2020 ◽  
Vol 21 (8) ◽  
pp. 3459-3467 ◽  
Author(s):  
Anaïs Marconnet ◽  
Baptiste Michon ◽  
Christel Le Bon ◽  
Fabrice Giusti ◽  
Christophe Tribet ◽  
...  

Author(s):  
L. M. Marshall

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.


Author(s):  
D.J. Benefiel ◽  
R.S. Weinstein

Intramembrane particles (IMP or MAP) are components of most biomembranes. They are visualized by freeze-fracture electron microscopy, and they probably represent replicas of integral membrane proteins. The presence of MAP in biomembranes has been extensively investigated but their detailed ultrastructure has been largely ignored. In this study, we have attempted to lay groundwork for a systematic evaluation of MAP ultrastructure. Using mathematical modeling methods, we have simulated the electron optical appearances of idealized globular proteins as they might be expected to appear in replicas under defined conditions. By comparing these images with the apearances of MAPs in replicas, we have attempted to evaluate dimensional and shape distortions that may be introduced by the freeze-fracture technique and further to deduce the actual shapes of integral membrane proteins from their freezefracture images.


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