Recognition of receptor lipopolysaccharides by spike G protein of bacteriophage phiX174.

The spike G protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged G protein (HisG). In the enzyme-linked plate assay, HisG bound specifically to lipopolysaccharides (LPSs) of the phiX174-sensitive strains, and did not bind to LPSs of the phiX174-insensitive strains. The truncated G protein obtained after trypsin digestion of HisG had the similar affinity to the LPSs to HisG, indicating that eight amino acid residues from the N-terminus are not essential to the binding with the LPSs.

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.

Specific interaction of fused H protein of bacteriophage phiX174 with receptor lipopolysaccharides.

The DNA fragment encoding the spike H protein of bacteriophage phiX174 was amplified by polymerase chain reaction. The fragment was sub-cloned into pQE-30 to yield pQE-H. The histidine-tagged H protein (HisH) was obtained from the cell extract of Escherichia coli M15 (pREP4) harboring pQE-H and purified by nickel chelating and anion-exchange chromatographies. HisH was shown to bind dose-dependently to the lipopolysaccharides (LPSs) isolated from phiX174-sensitive strains, E. coli C or Salmonella typhimurium TV119 (Ra mutant). In sharp contrast, HisH did not bind to the LPSs from insensitive strains, E. coli F583 (Rd2 mutant) or E. coli O111:B4 (smooth strain). Since the same selectivity was observed in the plaque counting assay for in vitro inactivation of phiX174, the spike H protein was shown to recognize receptor LPS.

Photoresponsive peptide and polypeptide systems: 10. Synthesis and reversible photochromism of azo aromatic poly(L-alpha,beta-diaminopropionic acid).

Poly-L-alpha,beta-diaminopropionic acid) having azo aromatic side chain was synthesized by the water-soluble carbodiimide procedure. The photochemical properties of the azo polypeptide poly[N beta-p-(phenylazo)benzoyl-L-alpha,beta-diaminopropionic acid] (PPABLDPA) was investigated by absorption and circular dichroism (c.d.) spectroscopy in hexafluoro-2-propanol (HFIP) and dimethylformamide. The photochromism of the absorption band in the visible and ultraviolet wavelength regions was found to be mostly reversible as a function of irradiation time at different wavelengths due to the photostationary state (88% trans)-cis photoisomerization of the azo aromatic moieties. The c.d. spectra exhibited two and three-stage photochromism on irradiation by light. The reversible photo-induced solubility change was also studied. On irradiation PPABLDPA is soluble under ultraviolet light (cis) and precipitates under visible light (88% trans) in HFIP-water. A discussion was presented that includes our previous results on this azo aromatic polylysine homologue series.