The interaction of theBacillus subtilisprotein 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 membranesin vivoandin 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 inEscherichia 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 membranesin vivoandin 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 inE. 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 genusBacillus. This information allowed us to discover a highly conserved Shine-Dalgarno sequence in the operonmstX-yugOthat is important for downstream translation of the potassium ion channelyugO.