title: Amino-acid residues involved in the expression of the activity of Escherichia coli TolC.
abstract: The Escherichia coli TolC, composed of 471 amino-acid residues, functions as a channel tunnel in the transport of various molecules across the outer membrane. We found previously that Leu-412, the 60th amino-acid residue from the carboxy terminal end, was crucial to the transport activity of TolC. Leu-412 is located in a domain which protrudes from the main body of TolC into the periplasm. Subsequent study indicated that the hydrophobicity generated by Leu-412 played an important role in the activity of TolC (H. Yamanaka, T. Nomura, N. Morisada, S. Shinoda, and K. Okamoto, Microb. Pathog. 33: 81-89, 2002). We predicted that other hydrophobic amino-acid residues around Leu-412 were also involved in the expression of the activity of TolC. To test this possibility, we substituted several hydrophobic residues around Leu-412, (Leu-3, Val-6, Leu-212, Leu-213, Leu-223, and Leu-224), with serine and examined the activity of these mutant TolCs. The result showed that Leu-3 is involved in the activity of TolC, but the other residues are not. The involvement of Leu-3 was confirmed by the residue deletion experiment. A subsequent point-mutational analysis of the residue showed that a hydrophobic side chain is required at position 3 for TolC to express its activity. As the distance between the alpha-carbons of Leu-3 and Leu-412 is just 7.45 angstroms, hydrophobic interaction between the two leucine residues might be involved in the activity of TolC.
authors.affiliation: Laboratory of Biochemistry, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, Japan. yama@ph.bunri-u.ac.jp
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Yamanaka
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Morisada
authors.forename: Masaya
authors.initials: M
authors.lastname: Miyano
authors.forename: Hideaki
authors.initials: H
authors.lastname: Tsuge
authors.forename: Sumio
authors.initials: S
authors.lastname: Shinoda
authors.forename: Eizo
authors.initials: E
authors.lastname: Takahashi
authors.forename: Keinosuke
authors.initials: K
authors.lastname: Okamoto
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003087:Colicins / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression / Q000502:physiology*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D021381:Protein Transport
chemical_list: D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli