Real-Time BIAcore Measurements of Escherichia coli Single-Stranded DNA Binding (SSB) Protein to Polydeoxythymidylic Acid Reveal Single-State Kinetics with Steric Cooperativity

The Pharmacia BlAcore biosensor was used to measure the real-time interaction of Escherichia coli SSB protein with poly(deoxythymidylic acid), (dT) 70 . The rationale for an experimental protocol for this type of binding reaction is presented. This approach may have general applicability in the design and analysis of BiAcore binding experiments. A chip is modified with three different surface densities of biotinylated oligonucleotide and a fourth channel is modified with only streptavidin. This allows examination of collected data for surface crowding effects, for flow limiting or reaction limiting binding, and for surface saturation. These observations are used to develop a mathematical model to describe the family of sensorgrams from a series of surface densities and protein concentrations with a single set of parameters. Each part of the model, as well as its relationship to the overall binding reaction, is described. The model presented is consistent with kinetic data for the association and dissociation rate constants for E. coli SSB and permits calculation of a thermodynamic binding constant. The concept of steric cooperativity is introduced to explain the observed increasing rate of binding as the surface binds more SSB protein.