Membrane proteins act as the gates, outposts and switches of cellular activity, performing numerous functions required for survival. These hydrophobic macromolecules insert into the complex environment of the lipid bilayer by adopting two types of fold: alpha-helical bundles and beta-barrels. In both structures, hydrophobic segments span the membrane and are separated by loop regions facing the water-head group-hydrophobic core interface - a chemically heterogenous environment. Membrane protein sequence must not only dictate the protein fold, but must also match the anisotropic environment of cellular membranes. Indeed, this solvent can also respond to the presence of a membrane protein, potentially affecting its fold. In order to evaluate the interplay between sequence and environment in membrane protein folding, systematic mutations in the environment-ambiguous loop region were used to probe the sequence of two membrane protein model transmembrane (TM) hairpins: TM 3 and 4 of CFTR, and the subunit c of the FoF1-ATP synthase. These helix-loop-helix constructs were used as minimal tertiary folding models in an array of biophysical and biochemical methods. First, the connection between the solvent and sequence changes was investigated in CFTR TM3/4 hairpins, with the finding that certain environments can be exquisitely sensitive to sequence changes upon solubilization of membrane proteins. Then, a sequence-dependent change from alpha-helix to beta-sheet in the membrane protein fold irrespective of the environment is described, where short-turn inducing sequence changes switch the secondary structure of CFTR TM3/4. Finally, this discovery was extended to the ancient membrane protein subunit c hairpins, hinting at a possible alpha-helical-to-beta-sheet conversion as a potential evolutionary impetus for the divergence of alpha-helical bundles to beta-barrel membrane proteins. The overall findings define a membrane protein folding paradigm in which both sequence and environment tailor the final fold of a membrane protein, whether alpha-helical or beta-sheet.Ph.D
