A Split Adenylate Kinase Sensor for Detecting Protein-Protein Interactions in Thermophiles

A variety of assays have been created for in vivo detection of protein-protein interactions within mesophilic organisms, but no methods have been generated for use within thermophiles, organisms that grow optimally at extreme temperatures. We describe the development of the first high-temperature protein-fragment complementation assay (HT-PCA) for analyzing protein-protein interactions within a thermophilic bacterium. Our assay is based on the complementation of a temperature-sensitive strain of Thermus thermophilus HB8 by a split adenylate kinase (AK) from Thermotoga neapolitana, which has a C156A mutation within its zinc-binding motif. We use this HT-PCA to analyze predicted interactions among thermophilic chemotaxis proteins and find that split AK function is enhanced upon fusion to proteins that interact. This assay will aid in creating genome-wide maps of thermophile protein-protein interactions, studying the effects of temperature on the strength of biomolecular interactions, and engineering oligomeric, thermostable nanomaterials for biotechnology, electronics, and medicine.