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

Abstract Background Vh ChiP 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 Vh ChiP 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 H 2 O and D 2 O. The BLM results revealed the significant contribution of hydrogen bonding to the binding of chitohexaose in the constriction zone of Vh ChiP. Replacing H 2 O as solvent by D 2 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 Trp 136 with the charged residues Asp or Arg. The on- and off-rates decreased up to 2.5-fold and 4-fold when Trp 136 was replaced by Arg, or 5-fold and 3-fold for Trp 136 replacement by Asp, respectively. Measuring the on-rate at different temperatures and for different channel mutants revealed the activation energy for chitohexaose entrance into Vh ChiP channel. Conclusions Hydrogen-bonds contribute to sugar permeation.