title: Mistic's membrane association and its assistance in overexpression of a human GPCR are independent processes.
abstract: The interaction of the Bacillus subtilis protein Mistic with the bacterial membrane and its role in promoting the overexpression of other membrane proteins are still matters of debate. In this study, we aimed to determine whether individual helical fragments of Mistic are sufficient for its interaction with membranes in vivo and in vitro. To this end, fragments encompassing each of Mistic's helical segments and combinations of them were produced as GFP-fusions, and their cellular localization was studied in Escherichia coli. Furthermore, peptides corresponding to the four helical fragments were synthesized by solid-phase peptide synthesis, and their ability to acquire secondary structure in a variety of lipids and detergents was studied by circular dichroism spectroscopy. Both types of experiments demonstrate that the third helical fragment of Mistic interacts only with LDAO micelles but does not partition into lipid bilayers. Interestingly, the other three helices interact with membranes in vivo and in vitro. Nevertheless, all of these short sequences can replace full-length Mistic as N-terminal fusions to achieve overexpression of a human G-protein-coupled receptor in E. coli, although with different effects on quantity and quality of the protein produced. A bioinformatic analysis of the Mistic family expanded the number of homologs from 4 to 20, including proteins outside the genus Bacillus. This information allowed us to discover a highly conserved Shine-Dalgarno sequence in the operon mstX-yugO that is important for downstream translation of the potassium ion channel yugO.
authors.affiliation: Department of Chemistry, University of Zürich, Switzerland.
authors.forename: Jacopo
authors.initials: J
authors.lastname: Marino
authors.forename: Natalie
authors.initials: N
authors.lastname: Bordag
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
authors.forename: Oliver
authors.initials: O
authors.lastname: Zerbe
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D008823:Micelles; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008823:Micelles; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: GFP-fusion; GPCR expression; Mistic; lipids; peptide-membrane interaction; yugO