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Methods Enzymol. 2019;617:1-27. doi: 10.1016/bs.mie.2018.12.001. Epub 2019 Jan 23.

Molecular snapshots of dynamic membrane-bound metabolons.

Author information

1
Plant Biochemistry Laboratory, Department of Plant and Environmental Science, University of Copenhagen, Copenhagen, Denmark; Center for Synthetic Biology "bioSYNergy", Copenhagen, Denmark; VILLUM Research Center "Plant Plasticity", Copenhagen, Denmark. Electronic address: jbassard@outlook.com.
2
Plant Biochemistry Laboratory, Department of Plant and Environmental Science, University of Copenhagen, Copenhagen, Denmark; Center for Synthetic Biology "bioSYNergy", Copenhagen, Denmark; VILLUM Research Center "Plant Plasticity", Copenhagen, Denmark. Electronic address: tola@plen.ku.dk.

Abstract

Numerous biosynthetic pathways have been shown to assemble at the surface of cellular membranes into efficient dynamic supramolecular assemblies termed metabolons. In response to environmental stimuli, metabolons assemble on-demand making them highly dynamic and fragile. This transient nature has previously hampered isolation and molecular characterization of dynamic metabolons. In contrast to conventional detergents, which tend to disrupt weak protein-protein interactions and remove lipids, the competence of a styrene maleic acid copolymer to carve out discrete lipid nanodisc from membranes offers immense potential for isolation of intact protein assemblies. Here, we present a method to extract the entire membrane-bound dhurrin pathway directly from microsomal fractions of the cereal Sorghum bicolor. This detergent-free nanodisc approach may be generally transposed for isolation of entire plant biosynthetic metabolons. This method provides a simple practical toolkit for the study of membrane protein complexes.

KEYWORDS:

Cytochromes P450; Detergent free; Dhurrin metabolon; Metabolon; Metabolon isolation; Microsome preparation; NADPH-dependent cytochrome P450 oxidoreductase; Styrene maleic acid copolymer; Styrene maleic acid lipid particles

PMID:
30784399
DOI:
10.1016/bs.mie.2018.12.001

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