Membrane transport proteins with complete replacement of transmembrane helices with polyalanine sequences remain functional.

Approximately 25% of all genome coding sequences correspond to membrane proteins, which perform varied and essential functions in cells. Eukaryotic integral membrane proteins are predominantly alpha-helical proteins that span the membrane several times. The most frequent approach to identifying transmembrane-helix amino acids essential for function is to substitute native residues, one at a time, with Cys or Ala (Cys- and Ala-scanning mutagenesis). Here, we present a new approach, in which complete transmembrane-helix native sequences are substituted with poly-Ala sequences. We show that the basic functional features of two dissimilar membrane proteins, which function as a channel and a pump, respectively, are maintained when certain individual alpha-helices are replaced with poly-Ala sequences. This approach ("helix-scanning mutagenesis") allows for rapid identification of helices containing residues essential for function and can be used as a primary helix-screening tool, followed by individual amino acid substitutions when specific helix poly-Ala replacements cause functional changes in the protein.