Complete and Reversible Chemical Denaturation of an α-Helical Membrane Protein

Abstract

The question of how an unordered polypeptide chain assumes its native, biologically active conformation is one of the greatest challenges in molecular biophysics and cell biology. This is particularly true for membrane proteins. Chemical denaturants such as urea have been used successfully for in vitro un- and refolding studies of soluble proteins and β-barrel membrane proteins. in stark contrast with these two protein classes, in vitro unfolding of α-helical membrane proteins by urea is often irreversible, and alternative denaturation assays using the harsh detergent sodium dodecyl sulphate suffer from a lack of a common reference state.Here we present the complete and reversible chemical denaturation of the bacterial α-helical membrane protein Mistic out of different micellar environments by urea. We applied multidimensional spectroscopy and techniques typically used in β-barrel membrane protein unfolding. Mistic unfolds reversibly following a two-state equilibrium that exhibits the same unfolded reference state. This allows for a direct comparison of the folding energetics in different membrane-mimetic systems and contributes to our understanding of how α-helical membrane proteins fold as compared with both β-barrel membrane proteins and water-soluble proteins.