Physiological evidence for an interaction between helix XI and helices I, II, and V in the melibiose carrier of Escherichia coli.
In a previous study 23 residues in helix XI of the cysteine-less melibiose carrier were changed individually to cysteine. Several of these cysteine mutants (K377C, A383C, F385C, L391C, G395C) had low transport activity and they were white on melibiose MacConkey fermentation plates. After several days of incubation of these white clones on melibiose MacConkey plates a rare red mutant appeared. The plasmid DNA was then isolated and sequenced. The two second site revertants from K377C were I22S and D59A. This change of aspartic acid to a neutral residue suggests that physiologically there is an interaction between K377 and D59 (possibly a salt bridge). The revertants from A383C were in positions 20 (F20L) and 22 (I22S and I22N). Revertants of F385C were intrahelical changes (I387M and A388G) and a change in C-terminal loop (R441C). Revertants of L391C were in helix I (I22N, I22T and D19E) and helix V (A152S). Revertants of G395C were in helix I (D19E and I22N). We suggest that there is an interaction between helix XI and helices I, II, and V and proximity between these helices.
Ding
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Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Wilson
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D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D008553:Melibiose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D017433:Protein Structure, Secondary; D012964:Sodium / Q000494:pharmacology; D027981:Symporters
D026901:Membrane Transport Proteins; D027981:Symporters; D002731:4-Chloromercuribenzenesulfonate; C021548:melibiose permease; D008553:Melibiose; D008094:Lithium; D012964:Sodium