Studies of dynamic protein-protein interactions in bacteria using Renilla luciferase complementation are undermined by nonspecific enzyme inhibition.
The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein interactions in live bacteria. As proof of concept and to develop a new tool for studies of bacterial chemotaxis, fragments of Renilla luciferase (Rluc) were fused to the chemotaxis-associated response regulator CheY3 and its phosphatase CheZ in the enteric pathogen Vibrio cholerae. Luciferase activity was dependent on the presence of both CheY3 and CheZ fusion proteins, demonstrating the specificity of the assay. Furthermore, enzyme activity was markedly reduced in V. cholerae chemotaxis mutants, suggesting that this approach can measure defects in chemotactic signaling. However, attempts to measure changes in dynamic CheY3-CheZ interactions in response to various chemoeffectors were undermined by nonspecific inhibition of the full-length luciferase. These observations reveal an unexpected limitation of split Rluc complementation that may have implications for existing data and highlight the need for great caution when evaluating small molecule effects on dynamic protein-protein interactions using the split luciferase technology.
Division of Infectious Diseases, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
Stavroula K
SK
Hatzios
Simon
S
Ringgaard
Brigid M
BM
Davis
Matthew K
MK
Waldor
D000818:Animals; D001419:Bacteria / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002633:Chemotaxis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D008156:Luciferases / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics; D000072236:Methyl-Accepting Chemotaxis Proteins; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D048790:Renilla; D014734:Vibrio cholerae / Q000235:genetics
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010446:Peptide Fragments; D008156:Luciferases