Dissection of discrete kinetic events in the binding of antibiotics and substrates to the galactose-H+ symport protein, GalP, of Escherichia coli.
GalP is the membrane protein responsible for H(+)-driven uptake of galactose into Escherichia coli. It is suggested to be the bacterial equivalent of the mammalian glucose transporter, GLUT1, since these proteins share sequence homology, recognise and transport similar substrates and are both inhibited by cytochalasin B and forskolin. The successful over-production of GalP to 35-55% of the total inner membrane protein of E. coli has allowed direct physical measurements on isolated membrane preparations. The binding of the antibiotics cytochalasin B and forskolin could be monitored from changes in the inherent fluorescence of GalP, enabling derivation of a kinetic mechanism describing the interaction between the ligands and GalP. The binding of sugars to GalP produces little or no change in the inherent fluorescence of the transporter. However, the binding of transported sugars to GalP produces a large increase in the fluorescence of 8-anilino-1-naphthalene sulphonate (ANS) excited via tryptophan residues. This has allowed a binding step, in addition to two putative translocation steps, to be measured. From all these studies a basic kinetic mechanism for the transport cycle under non-energised conditions has been derived. The case of genetical manipulation of the galP gene in E. coli has been exploited to mutate individual amino acid residues that are predicted to play a critical role in transport activity and/or the recognition of substrates and antibiotics. Investigation of these mutant proteins using the fluorescence measurements should elucidate the role of individual residues in the transport cycle as well as refine the current model.
Henderson
P J
PJ
Department of Biochemistry and Molecular Biology, University of Leeds, UK.
Martin
G E
GE
McDonald
T P
TP
Steel
A
A
Walmsley
A R
AR
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001693:Biological Transport, Active; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005576:Colforsin / Q000378:metabolism; D003571:Cytochalasin B / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005690:Galactose / Q000378:metabolism*; D005798:Genes, Bacterial; D051272:Glucose Transporter Type 1; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D000900:Anti-Bacterial Agents; C016242:AraF protein, E coli; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C495677:SLC2A1 protein, human; C023550:galactose-binding protein; D005576:Colforsin; D003571:Cytochalasin B; D005690:Galactose