The effect of modifications of the charged residues in the transmembrane helices on the transport activity of the melibiose carrier of Escherichia coli

The melibiose transport carrier of Escherichia coli (coded by melB gene) is a cotransport system which couples the transport of a-galactosides to prot ons, sodium, or lithium ions. The charged amino acid residues in membrane-s panning helices are of considerable interest because many of them have impo rtant function in substrate recognition. In most cases changing these charg ed residue to an uncharged residue (cysteine) results in total loss of acti vity. In this communication we describe experiments in which the cysteine s ubstitution for a charged residue was chemically changed by sulfhydryl reag ents (MTSEA and MTSET to restore a positive charge and MTSES a negative cha rge) or by iodoacetic acid or through oxidation by hydrogen peroxide so as to regain the original negative charge. In two cases (D55C and D124C) the r econstructed negative charges via the oxidation of the thiol to the sulfini c and/or sulfonic acid resulted in partial recovery of transport: D55C up t o 27% of the normal and D124C up to 4% of the normal in melibiose accumulat ion; D55C up to 36% of the normal and D124 up to 4.5% of the normal in down hill transport. Sulfhydryl reagents and iodoacetic acid failed to recover t ransport in all cases. We infer that the configurations of the charges as w ell as the structure of the side chains that carry them are critical in the maintenance of the transport. (C) 2001 Academic Press.