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-proteincoupled 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.

Conformational Dynamics Govern the Free-Energy Landscape of a Membrane-Interacting Protein

“…10 The protein contains 110 residues arranged into four α-helices. In spite of its polar, charged surface with a calculated pI of 4.5 and a net charge of −12 at pH 7, the helical bundle is tightly associated with membranes in vivo 9,11,12 and membrane-mimetic systems such as detergent micelles in vitro. 9,12−17 Lacking a signal recognition sequence, Mistic self-inserts into the bacterial membrane without the need for a translocon machinery.…”

“…The protein contains 110 residues arranged into four α-helices. In spite of its polar, charged surface with a calculated pI of 4.5 and a net charge of −12 at pH 7, the helical bundle is tightly associated with membranes in vivo ,, and membrane-mimetic systems such as detergent micelles in vitro. ,− Lacking a signal recognition sequence, Mistic self-inserts into the bacterial membrane without the need for a translocon machinery . Remarkably, polar and charged residues are distributed all over the protein rather than being clustered in distinct regions that could represent extramembraneous domains flanking a hydrophobic transmembrane domain, as is the case with canonical integral membrane proteins.…”

Design of an effective small expression tag to enhance GPCR production in E. coli‐based cell‐free and whole cell expression systems

“…To achieve efficient production of some GPCRs in various heterologous expression systems, including mammalian cells, yeast, E. coli , and CFPS system, several tags are known to be applicable to their N‐terminus (Fukutani et al, 2015; Lyukmanova et al, 2012; Marino et al, 2015). It is assumed that these tags do not significantly affect the function of GPCRs when attached to their N‐terminus.…”

Electrical signalling in prokaryotes and its convergence with quorum sensing in Bacillus

“…The role of YugO, a potassium efflux channel, was first investigated following the identification of the yugO locus downstream of the gene encoding the membrane associated protein Mistic (MstX), unique to the Bacillus genus. [ 16–18 ] The two genes are part of the same operon. As the MstX protein is associated with enhancing membrane protein expression, [ 19 ] Lundberg et al.…”