Thermodynamics of Mistic protein folding

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In vitro unfolding and refolding studies using chemical denaturants have contributed tremendously to our understanding of the folding thermodynamics and kinetics of water-soluble and β-barrel membrane proteins. However, this is not the case for α-helical membrane proteins, which constitute about one third of all proteins and more than half of all drug targets. We have identified the first example of an α-helical membrane protein of known structure that can be unfolded completely and reversibly by a denaturant: Mistic, a 110-residue, four-span membrane protein from Bacillus subtilis, dissociates from detergent micelles or lipid vesicles and assumes an unfolded monomeric state on titration with urea. Using site-directed mutagenesis, multidimensional spectroscopy, and Φ-value analysis, we aim to exploit this unique property to (i) establish a quantitative comparison of membrane-protein stability in different membrane-mimetic systems (micelles, bicelles, and vesicles); (ii) obtain insights at the amino acid level into the ability of this unusual membrane protein to self-insert spontaneously and in a translocon-independent way into lipid bilayers and biological membranes; and (iii) create a monomeric water-soluble Mistic variant that allows a direct comparison of protein folding between membrane-mimetic and aqueous environments.