Complete and Reversible Chemical Unfolding 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 folding 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 the lack of a common reference state. In line with this, we have recently demonstrated by NMR, CD, and fluorescence spectroscopy that urea is not able to completely abolish the secondary and tertiary structure of the α-helical membrane protein Mistic as long as the protein is solubilised in LDAO micelles. However, now we present the complete and reversible chemical unfolding of Mistic out of alkyl maltoside, cycloalkyl maltoside, and alkyl glucoside micelles. As revealed by automated CD 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 irrespective of the detergent used to solubilise the folded protein. The unfolded reference state contains virtually no secondary structure and tertiary contacts. 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 β-barrel membrane proteins and water-soluble proteins.