title: Determining the Topology of Membrane-Bound Proteins Using PEGylation.
abstract: Biochemical methods can help elucidate the membrane topology of hydrophobic membrane proteins where X-ray crystallography is difficult or impractical, providing important structural data. Here, we describe the method of PEGylation, which uses a cysteine-reactive molecule, maleimide polyethylene glycol (mPEG), to determine the cytosolic accessibility of introduced cysteine residues. This accessibility is visualized using Western blotting to detect a band shift that indicates cysteine labeling by mPEG. Using scanning cysteine mutagenesis, followed by PEGylation, one can map the accessibility of the introduced cysteines, hence inferring the membrane topology of the protein.We used PEGylation to determine the membrane topology of the sterol regulatory domain of a cholesterol synthesis enzyme, squalene monooxygenase, identifying that it is anchored to the membrane via a re-entrant loop.
authors.affiliation: School of Biotechnology and Biomolecular Sciences, Biological Sciences Building D26, The University of New South Wales, Sydney, NSW, 2052, Australia.
authors.forename: Vicky
authors.initials: V
authors.lastname: Howe
authors.affiliation: School of Biotechnology and Biomolecular Sciences, Biological Sciences Building D26, The University of New South Wales, Sydney, NSW, 2052, Australia. aj.brown@unsw.edu.au.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Brown
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D002784:Cholesterol / Q000096:biosynthesis / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D011092:Polyethylene Glycols / Q000737:chemistry*; D050603:Squalene Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011092:Polyethylene Glycols; D002784:Cholesterol; D050603:Squalene Monooxygenase; D003545:Cysteine
keywords: Membrane topology; Microsome; PEGylation; Squalene monooxygenase; Western blot