Information content of amino acid residues in putative helix VIII of the lac permease from Escherichia coli.
Mutants in putative helix VIII of lactose permease that retain the ability to accumulate lactose were created by cassette mutagenesis. A mutagenic insert encoding amino acid residues 259-278 was synthesized chemically by using reagents contaminated with 1% each of the other three bases and ligated into a KpnI/BclI site in the lacY gene in plasmid pGEM-4. Mutants that retain transport activity were selected by transforming a strain of Escherichia coli containing a wild-type lacZ gene, but deleted in lacY, with the mutant library and identifying colonies that transport lactose on indicator plates. Sequencing of the mutated region in lacY in 129 positive colonies reveals 43 single amino acid mutations at 26 sites and 26 multiple mutations. The variable amino acid positions are largely on one side of the putative alpha-helix, a stripe opposite Glu269. This mutable stripe of low information content is probably in contact with the membrane phospholipids.
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Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease