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

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

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/509141 doi: bioRxiv preprint allow crosslinking, which was followed by quenching in 1M ethanolamine, pH 8.5 for 300 seconds. Baseline was established in 1.8 mM Triton X-100 (diluted in water) over a period of 300 seconds. Sensors were then exposed to various OmpA-TM analytes (ranging from 1,000 nM to 7.8 nM) for 300 seconds to measure association. Dissociation was measured for 300 seconds by dipping the sensors into 1.8 mM Triton X-100. Data were reference-subtracted using the signal from crosslinked phage exposed only to 1.8 mM Triton X-100. Nonspecific binding was measured by exposing a sensor without tethered phage to the highest concentration of OmpA-TM S.flex and was found to be negligible. Data were fit using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA) and BiaEvaluation Software (GE Healthcare, USA). Experiments were performed in triplicate. Global fits were calculated from each set of experimental data, and overall there was relatively little binding variation between separate titrations.