Cysteine mutagenesis of the amino acid residues of transmembrane helix I in the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a sugar-cation cotransport sys tem that utilizes Na+, Li+: or H+. This membrane transport protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose car rier, cysteine has been substituted individually for amino acids 17-37, whi ch includes all of the residues in membrane helix I. The carriers with cyst eine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloromercuribenzenesulfonic aci d (PCMBS). Cysteine substitution caused loss of transport activity in six o f the mutants (G17C, K18C, D19C, Y32C, T34C, and D35C). PCMBS caused greate r than 50% inhibition in eleven mutants (F20C, A21C, I22C, G23C, I24C, V25C , Y26C, M27C, Y28C, M30C, and Y31C). We suggest that the residues whose cys teine derivatives were inhibited by PCMBS face the aqueous channel and that helix I is completely surrounded by aqueous environment. Second site rever tants were isolated from K18C and Y31C. The revertants were found to have m utations in helices I, IV, and VII.