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.
Highlights
VhChiP is a sugar uptake channel specific for chitohexaose
Generation of VhChiP Mutants—To confirm the functional importance of Trp136 in ion and sugar transport, we carried out site-directed mutagenesis to evaluate the impact of this residue in regulating ion conductance, binding affinity, and sugar permeability through VhChiP
The Western blot demonstrated that the purified VhChiP wild type (WT) and mutants all reacted strongly with the specific anti-VhChiP serum, whereas E. coli OmpN, which was often found to contaminate our recombinant VhChiP sample when the proteins were expressed in the porin-deficient E. coli BL21 (DE3) Omp8 Rosetta strain, was not recognized by the antibodies
Summary
VhChiP is a sugar uptake channel specific for chitohexaose. Significance: Chitin uptake by the highly virulent bacterium V. harveyi through VhChiP is dependent on hydrophobic interactions between the sugar molecule and the channel surface. We employed electrophysiological and biochemical approaches to demonstrate that Trp136, located at the mouth of the VhChiP pore, plays an essential role in controlling the channel’s ion conductivity, chitin affinity, and permeability. Liposome swelling assays confirmed that the Trp136 mutations decreased the rate of bulk chitohexaose permeation through the VhChiP channel. 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
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