PMID:8841135

Citation	He, MM, Sun, J and Kaback, HR (1996) Cysteine-scanning mutagenesis of transmembrane domain XII and the flanking periplasmic loop in the lactose permease of EScherichia coli. Biochemistry 35:12909-14
Abstract	Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain XII and the periplasmic loop between putative helices XI and XII (loop XI/XII) was replaced individually with Cys. Out of 34 mutants, 31 exhibit 60-100% or more of C-less activity, mutants Gly377-->Cys and Leu385-->Cys exhibit lower rates of transport but accumulate lactose about 60-70% as well as C-less, and mutant Leu400-->Cys exhibits < 20% of C-less activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to that of C-less with the exception of mutants Gly377-->Cys and Leu385-->Cys which are expressed about 40% as well as C-less and mutant Leu400-->Cys which is hardly detectable. When transferred to the wild-type background, however, mutant Leu400-->Cys is expressed normally and exhibits highly significant transport activity. Finally, each active Cys-replacement mutant was assayed for sensitivity to N-ethylmaleimide, and with three exceptions, the mutants are essentially unaffected by the alkylating agent. Mutants Val367-->Cys, Gly370-->Cys, and Tyr373-->Cys which are predicted to be immediately distal to helix XI in loop XI/XII are significantly inactivated. The periodicity observed suggests that the periplasmic end of transmembrane domain XI may extend to position 373. In the following paper [Voss, J., He, M. M., Hubbell, W. L., & Kaback, H. R. (1996) Biochemistry 35, 12915-12918], site-directed spin labeling of single-Cys mutants at positions 387-402 is used to demonstrate that transmembrane domain XII is in an alpha-helical conformation.
Links	PubMed Online version:10.1021/bi960876b
Keywords	Amino Acid Sequence; Biological Transport; Blotting, Western; Cell Membrane/enzymology; Cysteine/chemistry; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Ethylmaleimide/pharmacology; Lactose/metabolism; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Models, Molecular; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Protein Structure, Secondary; Symporters
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