Estimating loop-helix interfaces in a polytopic membrane protein by deletion analysis.
Insertions of amino acids into transmembrane helices of polytopic membrane proteins disrupt helix-helix interactions with loss of function, while insertions into loops have little effect on transmembrane helices and therefore little effect on activity [Braun, P., Persson, B., Kaback, H. R., and von Heijne, G. (1997) J. Biol. Chem. 272, 29566-29571]. Here the inverse approach, amino acid deletion, is utilized systematically to approximate loop-helix boundaries in the lactose permease of Escherichia coli. Starting with deletion mutants in the periplasmic loop between helices VII and VIII (loop VII/VIII), which has been defined by immunological analysis and nitroxide-scanning electron paramagnetic resonance spectroscopy, it is shown that mutants with single or multiple deletions in the central portion of the loop retain significant transport activity, while deletion of amino acid residues near the loop-helix boundaries or within the flanking helices leads to complete inactivation. Results consistent with hydropathy analysis are obtained with loops VI/VII, VIII/IX, and IX/X and the flanking helices. In contrast, deletion analysis of loops III/IV, IV/V, and V/VI and the flanking helices indicates that this region of the permease differs from hydropathy predictions. More specifically, evidence is presented supporting the contention that Glu126 and Arg144 which are charge paired and critical for substrate binding are within helices IV and V, respectively.
Wolin
C D
CD
Howard Hughes Medical Institute, Department of Physiology, Los Angeles, California 90025-1662, USA.
Kaback
H R
HR
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids / Q000138:chemical synthesis; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D027981:Symporters
D003062:Codon; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D027981:Symporters; D018698:Glutamic Acid; C021499:lactose permease; D001120:Arginine