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 mu tants (K377C, A383C, F385C, L391C, G395C) had low transport activity and th ey were white on melibiose MacConkey fermentation plates. After several day s of incubation of these white clones on melibiose MacConkey plates a rare red mutant appeared. The plasmid DNA was then isolated and sequenced. The t wo second site revertants from K377C were I22S and D59k This change of aspa rtic acid to a neutral residue suggests that physiologically there is an in teraction between K377 and D59 (possibly a salt bridge). The revertants fro m A383C were in positions 20 (F20L) and 22 (I22S and I22N). Revertants of F 385C 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) an d 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, an d V and proximity between these helices. (C) 2000 Academic Press.