A Chemical Strategy for Amphiphile Replacement in Membrane Protein Research

Abstract

Membrane mimics are indispensable tools in the structural and functional understanding of membrane proteins (MPs). Given stringent requirements of integral MP manipulations, amphiphile replacement is often required in sample preparation for various biophysical purposes. Current protocols generally rely on physical methodologies and rarely reach complete replacement. In comparison, we report herein a chemical alternative that facilitates the exhaustive exchange of membrane-mimicking systems for MP reconstitution. This method, named sacrifice-replacement strategy, was enabled by a class of chemically cleavable detergents (CCDs), derived from the disulfide incorporation in the traditional detergent n-dodecyl-β-d-maltopyranoside. The representative CCD behaved well in both solubilizing the diverse α-helical human G protein-coupled receptors and refolding of the β-barrel bacterial outer membrane protein X, and more importantly, it could also be readily degraded under mild conditions. By this means, the A2A adenosine receptor was successfully reconstituted into a series of commercial detergents for stabilization screening and nanodiscs for electron microscopy analysis. Featured by the simplicity and compatibility, this CCD-mediated strategy would later find more applications when being integrated in other biophysics studies.

Synthesis, Self-Assembly Properties, and Degradation Characterization of a Nonionic Photocleavable Azo-Sulfide Surfactant Family

“…Thus, the mAzo surfactants can serve as a cleavable alternative to common biological surfactants when this functionality is desirable ( e.g. , detergent exchange). , …”

Glycosyl disulfides: importance, synthesis and application to chemical and biological systems

“…6 In situ sacrifice-replacement strategy for reconstitution of mebrane proteins mediated by chemically cleavable detergents. 108 Reproduced by permission of ACS publications.…”

“…Even so, Tao and co-workers revived this proof-of-concept, and last year reported the successful use of glycosyl disulfides with hydrophobic aglycones as chemically cleavable detergents (CCDs) to purify and reconstitute MPs. 108 Essentially, they transformed the traditional detergent n-dodecyl-β-D-maltopyranoside (non-cleavable) into the respective disulfide as a CCD, which in turn behaved well in both solubilizing and refolding the protein A2R, also being readily degraded under mild conditions. Full cleavage and removal of CCD-2 allowed for the in situ reconstitution of the MPs into adequate commercial detergents for stabilisation screening, or into nanodiscs for electron microscopy analysis, under an approach that the authors termed sacrifice-replacement strategy (Fig.…”

Transition‐Linker Containing Detergents for Membrane Protein Studies

“…[6][7] The resulting protein detergent complex (PDC) could be applied directly, or after being reconstituted into other mimics, for subsequent biophysics studies. [8][9] Micelles, as well as PDCs, are in dynamic equilibrium with detergent monomers, rendering the hydrophobic domain of MPs exposed that destabilizes MPs and promotes aggregation. Stabilization of MPs is the foremost requisite for detergent evaluation.…”