Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein‐DNA binding affinities, nitrocellulose filter binding assays with 32P‐labeled DNA quantify Kd values from 10−12 to 10−8 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio koff/kon determines Kd. However, for lactose repressor protein (LacI) and an engineered repressor protein (“LLhF”) binding immobilized DNA, complicated kinetic curves precluded this analysis. Thus, we determined whether the amplitude of the BLI signal at equilibrium related linearly to the fraction of protein bound to DNA. A key question was the effective concentration of immobilized DNA. Equilibrium titration experiments with DNA concentrations below Kd (equilibrium binding regime) must be analyzed differently than those with DNA near or above Kd (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concentration was ~2 × 10−9 M. Although variation occurred among different lots of sensor tips, binding events with Kd ≥ 10−8 M should reliably be in the equilibrium binding regime. We also observed effects from multi‐valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amount of inducer sugars required to allosterically diminish protein‐DNA binding and to assess the affinity of fructose‐1‐kinase for the DNA‐LLhF complex. Overall, when experimental design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equilibrium titrations and thereby quantifying a variety of binding interactions.

SummaryIn E. coli, the master transcription regulator Cra regulates >100 genes in central metabolism by binding upstream DNA operator sequences. Genes encoding glycolytic enzymes are repressed, whereas those for gluconeogenesis and the citric acid cycle are activated. Cra-DNA binding is allosterically diminished by binding to either fructose-1-phosphate (F-1-P, generated upon fructose import) or fructose-1,6-bisphosphate (F-1,6-BP). F-1,6-BP is generated from F-1-P by the enzyme fructose-1-kinase (FruK) or from other sugars and is a key intermediate in glycolysis. Here, we report that Cra directly interacts with FruK to form a tight protein-protein complex. Further, growth assays with a fruK knockout strain show that FruK has a broader role in metabolism than its known role in fructose catabolism. Biochemical experiments show that F-1,6-BP binding enhances either the Cra/FruK interaction and/or CRA binding to DNA and that FruK can catalyze the reverse reaction of F-1,6-BP to F-1-P. Results were used to propose a model in which the Cra-FruK complex enhances activation of gluconeogenic genes. Finally, since FruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli central metabolism that occur in response to changing nutrients.