Probing Protein–Protein Interactions with Genetically Encoded Photoactivatable Cross-Linkers

Abstract

Fundamental to all living organisms is the ability of proteins to interact with other biological molecules at the right time and location, with the proper affinity, and to do so reversibly. One well-established technique to study protein interactions is chemical cross-linking, a process in which proteins in close spatial proximity are covalently tethered together. An emerging technology that overcomes many limitations of traditional cross-linking methods is one in which photoactivatable cross-linking noncanonical amino acids are genetically encoded into a protein of interest using the cell’s native translational machinery. These proteins can then be used to trap interacting biomolecules upon UV illumination. Here, we describe a method for the site-specific incorporation of photoactivatable cross-linking amino acids into fluorescently tagged proteins of interest in E. coli. Photo-cross-linking and analysis by SDS-PAGE using in-gel fluorescence detection, which provides rapid, highly sensitive, and specific detection of cross-linked adducts even in impure systems, are also described. An example expression and cross-linking experiment involving transmembrane signaling of a bacterial second messenger receptor system that controls biofilm formation is shown. All reagents needed to carry out these experiments are commercially available, and do not require special or unique technology to perform, making this method tractable to a broad community studying protein structure and function.