Kinetic Analysis of Bacteriophage Sf6 Binding to Outer Membrane Protein A Using Whole Virions

Natalia B. Hubbs; Mareena M. Whisby-Pitts; Jonathan L. McMurry

Temperature does not significantly change the kinetic parameters of Sf6 and OmpA binding. We purified the transmembrane domain of S. flexneri OmpA ("OmpA-TM S.flex " [23] ), confirmed we had functional OmpA-TMs by our previously reported assays [23, 25] ,and then measured the ability of OmpA-TMs to induce Sf6 genome ejection in vitro prior to performing BLI experiments. For all BLI experiments described herein, the ligand, Sf6, was immobilized on amine reactive (AR2G) sensors. OmpA-TMs reconstituted into detergent micelles were used as analytes. To ensure that sodium acetate, pH 4.0 buffer (a low pH buffer in which phage are not typically stored, but which was necessary for tethering to sensors) had no effect on the phage, we monitored the titer of the phage stock over time, comparing it to phage stored in phage dilution buffer, pH 7.6, and found no significant differences. Moreover, we tested the ability of OmpA-All rights reserved. No reuse allowed without permission.