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Research Themes Membrane proteins

Gentle membrane protein extraction

SBKB [doi:10.1038/sbkb.2011.59]
Technical Highlight - January 2012
Short description: Noncovalent chelation of nickel-doped PEG polymers to His-tagged integral membrane proteins permits the solubilization of functional proteins in low-detergent or detergent-free aqueous buffer.

Schematic of the reversible method used to stabilize solubilized His-tagged membrane proteins. Figure courtesy of Jessica Canseco.

Many proteins are embedded in cellular membranes. These integral membrane proteins (IMPs) include several categories of proteins of fundamental as well as medical importance, such as ion channels, G protein-coupled receptors and receptor tyrosine kinases. But IMPs are challenging to study. Structural and structure-function studies often require protein extraction from the membrane. This is most commonly accomplished by detergent solubilization, which is notoriously prone to causing protein denaturation and destabilization. Even when IMPs remain stable in detergent, solubilization can disrupt their native structure.

Alternative methods for membrane protein extraction do exist. IMPs have been solubilized with polyacrylamide-based surfactants (amphipols) to shield the hydrophobic transmembrane domains or stabilized by covalent modification with polyethylene glycol (PEG). But existing methods still have drawbacks: even in the best cases, extensive optimization is typically needed to find conditions that yield functional protein.

Lesley and colleagues (PSI JCSG) now build on the notion that 'PEGylation' can stabilize IMPs. Instead of covalently modifying IMPs with PEG, as has been previously reported, the researchers chelated nickel-doped tris-NTA monomethoxy (m)PEG polymers to His-tagged proteins. This has the benefit of not requiring extensive optimization or harsh conditions and is reversible with imidazole and EGTA. The researchers synthesized mPEG-NTA3(Ni2+) polymers of different sizes and chelated them to five different detergent-solubilized IMPs. They subsequently dialysed away the detergent and tested for protein stability. Depending on the polymer and the protein used, IMPs chelated with mPEG-NTA3(Ni2+) were stable either with minimal detergent or even in entirely detergent-free aqueous buffer.

Based on circular dichroism spectroscopic studies, the authors concluded that the mPEG-chelated IMPs had similar secondary structure to native and to amphipol-stabilized proteins. What is more, kinase assays on diacyl glycerol kinase showed that the extraction method could yield functional protein. Finally, imidazole and EGTA could reverse the chelation and return the IMPs to the detergent phase. This method should enable structural studies on several members of this important class of proteins.

Natalie de Souza

References

  1. T.K. Janaratne et al. Solubilization of native integral membrane proteins in aqueous buffer by noncovalent chelation with monomethoxy poly (ethylene glycol) (mPEG) polymers.
    Bioconjug Chem. 22, 1513-1518 (2011). doi:10.1021/bc200019x

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