In our ongoing studies of non-conserved amino acid positions, we have engineered a large number of LacI/GalR transcriptional regulators via domain recombination and amino acid substitution. To understand how these changes modified repressor function, we desired a quick and robust analytical method to quantify protein-DNA interactions. Existing equilibrium methods for measuring affinities are time consuming and are often limited to a discrete range of Kd values. Thus, we considered whether Biolayer Interferometry (BLI) was appropriate for our purpose. The known method of calculating Kd using BLI is to determine the ratio koff/kon. However, the protein-DNA kinetic curves in BLI assays were multiphasic and could not be meaningfully fit using a simple exponential phase. Nevertheless, we noted that as the binding reaction reached equilibrium, the BLI amplitude appeared sensitive to experiments differences known to alter affinity. Therefore, we next evaluated the strength and limitations of using BLI in the equilibrium regime to obtain apparent Kd values. Here, we present the results from assessing a range of experimental designs, including varied DNA sequences, varied protein concentrations, and varied concentrations of effector small molecules (which alter protein-DNA binding). With this quick, label free method, we have successfully and quickly assessed the apparent binding affinities of protein-DNA complexes.

Using of bio-layer interferometry to quantify protein-DNA interactions

2019 Fall National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.