title: Chitoporin from the Marine Bacterium Vibrio harveyi: PROBING THE ESSENTIAL ROLES OF TRP136 AT THE SURFACE OF THE CONSTRICTION ZONE.
abstract: VhChiP is a sugar-specific porin present in the outer membrane of the marine bacterium Vibrio harveyi. VhChiP is responsible for the uptake of chitin oligosaccharides, with particular selectivity for chitohexaose. In this study, we employed electrophysiological and biochemical approaches to demonstrate that Trp(136), located at the mouth of the VhChiP pore, plays an essential role in controlling the channel's ion conductivity, chitin affinity, and permeability. Kinetic analysis of sugar translocation obtained from single channel recordings indicated that the Trp(136) mutations W136A, W136D, W136R, and W136F considerably reduce the binding affinity of the protein channel for its best substrate, chitohexaose. Liposome swelling assays confirmed that the Trp(136) mutations decreased the rate of bulk chitohexaose permeation through the VhChiP channel. Notably, all of the mutants show increases in the off-rate for chitohexaose of up to 20-fold compared with that of the native channel. Furthermore, the cation/anion permeability ratio Pc/Pa is decreased in the W136R mutant and increased in the W136D mutant. This demonstrates that the negatively charged surface at the interior of the protein lumen preferentially attracts cationic species, leading to the cation selectivity of this trimeric channel.
authors.affiliation: From the Biochemistry-Electrochemistry Research Unit, the School of Biochemistry, and.
authors.forename: Watcharin
authors.initials: W
authors.lastname: Chumjan
authors.affiliation: the Department of Life Sciences and Chemistry, Jacobs University Bremen, D-28759 Bremen, Germany m.winterhalter@jacobs-university.de.
authors.forename: Mathias
authors.initials: M
authors.lastname: Winterhalter
authors.affiliation: From the Biochemistry-Electrochemistry Research Unit, the School of Chemistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand and.
authors.forename: Albert
authors.initials: A
authors.lastname: Schulte
authors.affiliation: the Department of Life Sciences and Chemistry, Jacobs University Bremen, D-28759 Bremen, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Benz
authors.affiliation: From the Biochemistry-Electrochemistry Research Unit, the School of Biochemistry, and wipa@sut.ac.th.
authors.forename: Wipa
authors.initials: W
authors.lastname: Suginta
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010539:Permeability; D011485:Protein Binding; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D014733:Vibrio
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008081:Liposomes; D009844:Oligosaccharides; C050523:chitohexaose; D014364:Tryptophan
keywords: carbohydrate-binding protein; chitin; lipid bilayer; single molecule biophysics; sugar transport