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 protons, sodium, or lithium ions. The charged amino acid residues in membrane-spanning helices are of considerable interest because many of them have important function in substrate recognition. In most cases changing these charged residue to an uncharged residue (cysteine) results in total loss of activity. In this communication we describe experiments in which the cysteine substitution for a charged residue was chemically changed by sulfhydryl reagents (MTSEA and MTSET to restore a positive charge and MTSES a negative charge) 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 reconstructed negative charges via the oxidation of the thiol to the sulfinic and/or sulfonic acid resulted in partial recovery of transport: D55C up to 27% of the normal and D124C up to 4% of the normal in melibiose accumulation; D55C up to 36% of the normal and D124 up to 4.5% of the normal in downhill transport. Sulfhydryl reagents and iodoacetic acid failed to recover transport in all cases. We infer that the configurations of the charges as well as the structure of the side chains that carry them are critical in the maintenance of the transport.
Ding
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PZ
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Wilson
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TH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008698:Mesylates / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D027981:Symporters
D026901:Membrane Transport Proteins; D008698:Mesylates; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; C093171:methanethiosulfonate ethylammonium; C109153:(2-sulfonatoethyl)methanethiosulfonate; C021548:melibiose permease; D008553:Melibiose; D005020:Ethyl Methanesulfonate; D006861:Hydrogen Peroxide; D003545:Cysteine