Functional estimation of loop-helix boundaries in the lactose permease of Escherichia coli by single amino acid deletion analysis.
Mutants with single amino acid deletions in the loops of lactose permease retain activity, while mutants with single deletions in transmembrane helices are inactive, and the loop--helix boundaries of helices IV, V, VII, VIII, and IX have been approximated functionally by the systematic deletion of single residues [Wolin, C. D., and Kaback, H. R. (1999) Biochemistry 38, 8590-8597]. The experimental approach is applied here to the remainder of the permease. Periplasmic and cytoplasmic loop-helix boundaries for helices I, II, X, XI, and XII and the cytoplasmic boundary of helix III are in reasonable agreement with structural predictions. In contrast, the periplasmic end of helix III appears to be five to eight residues further into the transmembrane domain than predicted. Taken together with the previous findings, the analysis estimates that 11 of the 12 transmembrane helices have an average length of 21 residues. Surprisingly, deletion analysis of loop V/VI, helix VI, and loop VI/VII does not yield an activity profile typical of the rest of the protein, as individual deletion of only three residues in this region abolishes activity. Thus, transmembrane domain VI which is probably on the periphery of the 12-helix bundle may make few functionally important contacts.
Wolin
C D
CD
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90025-1662,USA.
Kaback
H R
HR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D001693:Biological Transport, Active / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D017434:Protein Structure, Tertiary / Q000235:genetics; D027981:Symporters
D000596:Amino Acids; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D027981:Symporters; D010649:Phenylalanine; C021499:lactose permease