Characterization of the binding of spike H protein of bacteriophage phiX174 with receptor lipopolysaccharides.
The spike H protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of phiX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs. In sharp contrast, HisH bound weakly to the LPSs of phiX174-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 fluorescence 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 shorter 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 microM, and the Gibbs free energy change DeltaG(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 plot, the standard enthalpy change DeltaH(0) and the entropy change DeltaS(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-driven reaction.
Inagaki
M
M
Department of Bioscience, Faculty of Bioresources, Mie University, Kamihama, Tsu, Japan. inagaki@bio.mie-u.ac.jp
Tanaka
A
A
Suzuki
R
R
Wakashima
H
H
Kawaura
T
T
Karita
S
S
Nishikawa
S
S
Kashimura
N
N
D010584:Bacteriophage phi X 174 / Q000378:metabolism*; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012486:Salmonella typhimurium / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D008070:Lipopolysaccharides; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins