Polar Interactions Trump Hydrophobicity in Stabilizing a Membrane-Interacting Protein

Abstract

The vast majority of membrane-bound proteins consist of bundles of hydrophobic α-helical transmembrane domains. How these proteins adopt their native, biologically active structures in the apolar milieu of a membrane is increasingly well understood. However, it is still under debate to what extent polar interactions contribute to membrane-protein stability. For example, the membrane-interacting protein Mistic exhibits a net charge of −12 at pH 7 but, in NMR experiments, displays a concentric ring of interactions with apolar detergent tails typical of more hydrophobic membrane proteins. Moreover, as a fusion tag, Mistic supports the production and membrane insertion of integral membrane proteins in Escherichia coli. Thus, in spite of its unusually polar surface, Mistic in many aspects behaves like a conventional membrane protein.Here, we demonstrate that, upon addition of urea, Mistic unfolds reversibly form detergent micelles following a two-state equilibrium and exhibits the same unfolded reference state irrespective of the detergent used. Unfolding titrations from alkyl maltoside detergents revealed that alkyl tails of 12 carbon atoms preserve the conformational stability of the protein best. However, on top of such hydrophobic interactions, we discovered that zwitter-ionic and charged detergent head groups render Mistic extraordinarily resistant against unfolding, so that the protein largely retains its secondary structure even at urea concentrations higher than 7 M. Notably, for the application of Mistic as a fusion tag, polar or charged detergents have been found to be very successful in solubilizing the protein, which is in excellent agreement with our in vitro stability data. Thus, Mistic represents a unique model system to quantitatively assess the contribution of polar interactions to membrane-protein folding and extends the established view that hydrophobic interactions are the single most important contributors to the stability of membrane-interacting proteins.