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.
Bao
Xiaoyong
X
Department of Neuroscience and Cell Biology and the Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas 77555-0437, USA.
Chen
Yongyue
Y
Lee
Sung Haeng
SH
Lee
Sung Chang
SC
Reuss
Luis
L
Altenberg
Guillermo A
GA
D000409:Alanine; D000595:Amino Acid Sequence; D018031:Connexin 43 / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine; D017629:Gap Junctions / Q000502:physiology; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D010455:Peptides; D010957:Plasmids; D017433:Protein Structure, Secondary
D000409:Alanine; D018031:Connexin 43; D003545:Cysteine; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010455:Peptides; C019529:polyalanine; C421109:multidrug resistance-associated protein 1