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

BackgroundVhChiP 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.MethodsVhChiP 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.ResultsThe role of hydrogen-bonding in sugar binding was investigated by comparing channel occlusion by chitohexaose in buffers containing H₂O and D₂O. The BLM results revealed the significant contribution of hydrogen bonding to the binding of chitohexaose in the constriction zone of VhChiP. Replacing H₂O as solvent by D₂O 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.ConclusionsHydrogen-bonds contribute to sugar permeation.