Polymerization of Salmonella Flagellin in Water and Deuterium Oxide Media

For polymerization of Salmonella flagellin into flagellar filaments, a D2O medium containing 0.15 m NaCl and 0.01 M phosphate buffer (pD 7.4) is more favorable thaa an H2O medium containing 0.15M NaCl and 0.01 M phosphate buffer (pH 7.0). Evidence for this was obtained from kinetic and equilibrium experiments carried out by viscosity measurements. Flagellin polymerization is known to consist of two steps, nucleation and elongation. It was found that when a D2O medium contained monomeric flagellin at concentrations higher than 10mg/ml, nucleation occurred rapidly, while this rarely happened in an H2O medium. In addition, elongation of short polymers in the presence of a particular concentration of monomer became faster when D2O medium replaced H2O medium. Polymers (flagellar filaments) produced in the two kinds of media were compared by circular dichroism (CD) measurements and electron microscope observations: They were found to be indistinguishable. The overall monomer-polymer equilibrium shifts towards monomer on raising the temperature. It was shown that the equilibrium temperature for a mixture of given concentrations of monomer and polymer was raised by several degrees when the medium was changed from H2O to D2O. At 25°, monomers in H2O and D2O media had very similar CD profiles, indicating that their conformations were similar, if not completely identical. However, the conformation in D2O medium was shown to be more heat-stable than that in H2O medium. The experimental results support the current view that when proteins are transferred to DTO media, intra- and intermolec-ular hydrophobic bonds and or hydrogen bonds are strengthened.