Amino acid substitution in the lactose carrier protein with the use of amber suppressors.
Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane alpha helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first alpha helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is concluded that Trp-33 may be involved directly or indirectly in sugar recognition.
Huang
A M
AM
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02215.
Lee
J I
JI
King
S C
SC
Wilson
T H
TH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins; D007150:Immunohistochemistry; D007785:Lactose / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008756:Methylgalactosides / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D013489:Suppression, Genetic; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism
D000596:Amino Acids; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides; C009468:thiomethylgalactoside; C021499:lactose permease; D008553:Melibiose; D007785:Lactose