Characterization of the binding of spike H protein of bacteriophage phi X74 with receptor lipopolysaccharides

The spike H protein of bacteriophage phi X174 was prepared as a hexa histid ine-tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found t o bind specifically to the lipopolysaccharides (LPSs) of phi X174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, havin g the complete oligosaccharide sequence of the R-core on the LPSs, In sharp contrast, HisH bound weakly to the LPSs of phi X174-insensitive strains, i .e. E. coli F583 (Rd(2)) lacking some terminal saccharides and E. coli O111 :B4 (smooth strain) having additional O-repeats on the R-core, The fluoresc ence spectra of HisH changed dose-dependently in the case of the LPS of E. coli C, the intensity increasing and the emission peak shifting to the shor ter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS, The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant K-d, 7.02 +/- 0.37 mu M, a nd the Gibbs free energy change Delta G(0), -29.1 kJ mol(-1) (at 22 degrees C, pH 7.4), Based on the temperature dependence of K-d in a van't Hoff plo t, the standard enthalpy change Delta H-0 and the entropy change Delta S-0 were calculated to be +23.7 kJ mol(-1) and 179 J mol(-1) K-1 at 22 degrees C, respectively, and this binding was thereby concluded to be an entropy-dr iven reaction.