Alanine insertion scanning mutagenesis of lactose permease transmembrane helices.
A priori, single residue insertions into transmembrane helices are expected to be highly disruptive to protein structure and function. We have carried out a systematic analysis of the phenotypes associated with Ala insertions into transmembrane helices in lactose permease, a multispanning Escherichia coli inner membrane protein. Insertion of alanine into the center of 7 transmembrane helices was found to abolish stable integration of lactose permease into the membrane or uphill lactose transport. A more detailed Ala insertion scan was made of transmembrane helix III. The results pin-point a central region of approximately 2 helical turns that is crucial for lactose permease stability and/or activity. A Trp scan in this region identified 2 residues essential for lactose permease stability. From these results, it appears that transmembrane helices have differential sensitivities to single residue insertions and that such mutations may be useful for identifying structurally and/or functionally important helix segments.
Braun
P
P
Department of Biochemistry, University of Stockholm, S-106 91 Stockholm, Sweden.
Persson
B
B
Kaback
H R
HR
von Heijne
G
G
D000409:Alanine / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins; D007785:Lactose / Q000493:pharmacokinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016254:Mutagenesis, Insertional; D017433:Protein Structure, Secondary; D027981:Symporters; D014364:Tryptophan / Q000235:genetics
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D014364:Tryptophan; C021499:lactose permease; D007785:Lactose; D000409:Alanine