POSTERS - BLAST X - University of Utah

BLAST X Poster #43
APPLICATION OF BIOLAYER INTERFEROMETRY TO UNDERSTANDING INTERACTIONS
AMONG SALMONELLA ENTERICA FLAGELLAR EXPORT APPARATUS PROTEINS
Jonathan L. McMurry
Department of Chemistry & Biochemistry, Kennesaw State University, Kennesaw, GA
Biolayer interferometry (BLI) is an emerging optical biosensing technology for the
analysis of dynamic biomolecular interactions. Measurements are made of changes in the
interference pattern of white light reflected off of two surfaces, one of which possesses a layer
of immobilized protein (or other molecule) and the other an internal reference. Binding of
second protein to the immobilized one results in a change in distance between the two surfaces
and thus a shift in wavelength of the interference pattern. Kinetic and affinity constants can thus
be determined in real-time, without attainment of equilibrium, utilizing small, recoverable
quantities of label-free biomolecules. Using a commercial BLI biosensor, interactions among
proteins involved in flagellar export in Salmonella enterica were investigated. Among
interactions measured were those between two proteins involved in specificity switching, FliK
and FlhB. FliK binding to wild-type FlhB and two variants (N269A and P270A) surprisingly
evidenced similar kinetic profiles with submicromolar KDs resulting from fast-on and fast-off rate
constants. Other interactions between export proteins, e.g., FliH-FliI, FliH-FliJ, demonstrated
stable, low kd binding, as expected. Additional efforts include screening for interactions too
weak to allow for copurification or other traditional binding assays, attempts to demonstrate
interactions between export proteins and substrates and analysis of more-than-pairwise
interactions. Further application of BLI to the investigation of flagellar export protein dynamics
will allow for better understanding of the mechanism of export.
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