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

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

We purified various OmpA-TMs: one from E. coli, "OmpA-TM E.coli " and four that deviated by single amino acid substitutions from the native S. flexneri sequence (D66A, N67E, P111E, and N155E). These variants were chosen to represent a broad range of phenotypes [23] . Here, we measured the kinetics of Sf6 binding to these various receptor types using BLI and calculated the kinetic and affinity constants for each ( Figure 3 , and Table 2 ). Again, some complexity was evident but a simple 1:1 binding model fit the data better than two-state parallel or conformational change models. The only exception was the lowest analyte concentration for N67E ( Figure 3 , magenta line), for which global fits did not converge, and was therefore eliminated from the analysis. The calculated parameters were consistent with fast-on and slowoff kinetics. OmpA-TM E.coli and all S.flexneri OmpA-TM variants bound Sf6 with nM affinity; the K D s ranged between 6.9 and 65.4 nM. The kinetic parameters of binding for the variants differed only slightly when compared to those of OmpA-TM S.flex . Moreover, the small differences observed do not correspond to the phenotypes previously reported [23] . For example,