Quantifying protein-protein interactions of the acyl carrier protein with solvatochromic probes.

Protein-protein interactions (PPIs) are universal to life and their study and understanding is critical to drug discovery and bioengineering efforts. Historically, X-ray crystallography, isothermal titration calorimetry and other biophysical methods have been used to study PPIs, but can be costly and are low throughput, hindering progress towards rapid evaluation of these interactions. Recent interest in targeting PPIs and in engineering biosynthetic pathways in which PPIs play a critical role has driven innovation in their evaluation but a universal screen is still needed. One of the best characterized systems relying upon PPIs is Escherichia coli type II fatty acid biosynthesis in which the central acyl carrier protein (EcACP) shuttles substrates to a series of partner enzymes. Here we present a method by which EcACP is labeled with a solvatochromic dye, 4-DMN, and then allowed to interact with its various partner enzymes. Upon interaction, there is a large increase in fluorescence intensity which is easily monitored via fluorometer or plate reader. This method is useful in the study of known PPI, hypothetical PPI and in evaluation of inhibitors of both partner enzyme active site and of the PPI itself.