Effects of H-bonds on sugar binding to chitoporin from Vibrio harveyi.

BACKGROUND: VhChiP is a sugar-specific-porin present in the outer membrane of the marine bacterium Vibrio harveyi and responsible for chitin uptake, with a high selectivity for chitohexaose.

METHODS: VhChiP and its mutants were expressed and purified from BL21 (DE3) Omp8 Rosetta strain. After reconstitution into planar lipid bilayers, the ion current fluctuations caused by chitohexaose entering the channel were measured in deuterium oxide and in water.

RESULTS: The role of hydrogen-bonding in sugar binding was investigated by comparing channel occlusion by chitohexaose in buffers containing H2 O and D2 O. The BLM results revealed the significant contribution of hydrogen bonding to the binding of chitohexaose in the constriction zone of VhChiP. Replacing H2 O as solvent by D2 O significantly decreased the on- and off-rates of sugar penetration into the channel. The importance of hydrogen bonding inside the channel was more noticeable when the hydrophobicity of the constriction zone was diminished by replacing Trp136 with the charged residues Asp or Arg. The on- and off-rates decreased up to 2.5-fold and 4-fold when Trp136 was replaced by Arg, or 5-fold and 3-fold for Trp136 replacement by Asp, respectively. Measuring the on-rate at different temperatures and for different channel mutants revealed the activation energy for chitohexaose entrance into VhChiP channel.

CONCLUSIONS: Hydrogen-bonds contribute to sugar permeation.