Helix packing in the sucrose permease of Escherichia coli: properties of engineered charge pairs between helices VII and XI.
Of four putative intramembrane charge pairs in lactose permease, only three are conserved in the homologous sucrose permease of Escherichia coli [Bockmann, J., Heuel, H., & Lengeler, J. W. (1992) Mol. Gen. Genet. 235, 22-32]. The missing charge pair was introduced into wild-type sucrose permease by site-directed mutagenesis of Asn234 (helix VII) and Ser356 (helix XI). Individual replacement of either residue with a charged amino acid abolishes active sucrose transport with the exception of the Asn234-->Asp mutant. However, simultaneous replacement of Asn234 with Asp or Glu and Ser356 with Arg or Lys results in high activity. Thus, an acidic residue at position 234 rescues the activity of the Ser356-->Arg or Ser356-->Lys mutant, and a basic residue at position 356 rescues the activity of the Asn234-->Glu mutant. Furthermore, when expressed at a relatively low rate, the double mutant Asn234-->Asp/Ser356-->Arg is present in the membrane in a significantly greater amount than wild-type, suggesting that the charge pair improves insertion of sucrose permease into the membrane. The results indicate that helices VII and XI of sucrose permease are in close proximity and that a charge pair interaction can be established between residues 234 (helix VII) and 356 (helix XI). However, interchange of the acidic residue at position 234 with the basic residue at position 356 abolishes sucrose transport.(ABSTRACT TRUNCATED AT 250 WORDS)
Frillingos
S
S
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Sahin-Tóth
M
M
Lengeler
J W
JW
Kaback
H R
HR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D003062:Codon; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism
D003062:Codon; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C056379:cscB protein, E coli; D013395:Sucrose