Most membrane proteins are permanently attached to lipid bilayers through hydrophobic transmembrane helices, whose topogenesis requires sophisticated insertion machineries.1 By contrast, only a few self-inserting integral membrane proteins are known, and it has remained unclear how these proteins are associated with the bilayer in the absence of canonical transmembrane domains. A puzzling example is provided by the self-inserting protein Mistic (MstX), which has attracted attention as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins. Mistic combines properties of both soluble as well as more typical membrane proteins. Although it contains numerous polar and charged residues and lacks characteristic membrane-interaction motifs such as amphipathic helices, it is tightly bound to membranes in vivo or membrane-mimetic systems in vitro.2 Using optical and NMR spectroscopy, we have demonstrated that Mistic can be unfolded completely and reversibly by urea.3 Here, we exploit this finding to dissect the contributions of polar and nonpolar interactions to its conformational stability. Unfolding from nonionic detergents revealed that protein stability steeply depends on hydrophobic micelle diameter, as expected for an integral membrane protein. However, headgroup chemistry was found to exert an even stronger influence, as polar interactions render the protein resistant against unfolding and can even induce a shift from two-state to one-state (“downhill”) folding. Taken together, these results shed light on the molecular determinants governing protein folding and stability in the complex environment of a hydrophilic/hydrophobic interface and rationalise the suitability of various detergents for the extraction and solubilisation of membrane proteins.