The preparation of samples exhibiting stable and natively-folded structures that retain the function of the membrane proteins is crucial for investigation of high-resolution structures and dynamics of membrane proteins.
Detergents and micelles, as traditional membrane mimetics, are continued to be used in the structural studies, mainly the X-ray crystallography studies. However, it is known that detergents destabilize protein structures and disrupt their functions. Bicelles, although considered to be better membrane mimetic systems, have also been shown to distort protein structures and functions due to the diffusion of the detergent molecules from the bicelle rim to the planar lipid bilayer region.
Dr. Bankala Krishnarjuna’s team published an article titled “Detergent-free extraction, reconstitution and characterization of membrane-anchored cytochrome-b5 in native lipids” in Chemical Communications, introducing a novel method for membrane protein extraction without destabilizing protein structures (Chemical Communications, 2020). In this study, the researchers demonstrated the feasibility of a direct extraction of a B16 kDa cytochrome-b5 from E. coli using a synthetic styrene-co-maleic acid-ethanolamine (SMA-EA) polymer and reconstituting the cytochrome-b5 protein in polymer nanodiscs along with native lipids from E. coli.
A schematic representation of the direct extraction of a 16 kDa rabbit cytochrome-b5 in native E. coli lipids using a negatively charged SMA-EA polymer. + and - indicate the net charge of a membrane protein. SEC: size-exclusion chromatography.
The result data of this research revealed that the directly extracted 15N-labelled cytochrome-b5 was of high purity as indicated by the 2D 1H/15N TROSY-HSQC NMR spectrum, which also demonstrated the correctly folded state of the protein and the feasibility to carry out structural and dynamical measurements using the well-established multidimensional solution NMR techniques.
Is extraction using synthetic polymers a best way for preparing membrane protein samples? Leave your thoughts in the comment section below to win a canvas bag, hoodie or $20 Amazon gift card! You can also have a chance to get one of those gifts by taking the short quiz below from Dr. Bankala regarding membrane protein extraction.
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Q1: How many membrane protein structures have been reported in the Protein Data Bank? (1 mark)
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Q2: Is it possible to extract the native lipids associated with a specific membrane protein in the cell membrane? (2 marks)
Q3: Which of the following can achieve 100% detergent-free extraction of membrane proteins? (3 marks)
Q4: Are membrane proteins extracted using synthetic polymers suitable for high-resolution structural studies by NMR (4 marks)?
Q5: Is the charge of the polymer used a limitation for achieving membrane protein extraction and reconstitution? (5 marks)