A selection that reports on protein-protein interactions within a thermophilic bacterium.
Many proteins can be split into fragments that exhibit enhanced function upon fusion to interacting proteins. While this strategy has been widely used to create protein-fragment complementation assays (PCAs) for discovering protein-protein interactions within mesophilic organisms, similar assays have not yet been developed for studying natural and engineered protein complexes at the temperatures where thermophilic microbes grow. We describe the development of a selection for protein-protein interactions within Thermus thermophilus that is based upon growth complementation by fragments of Thermotoga neapolitana adenylate kinase (AK(Tn)). Complementation studies with an engineered thermophile (PQN1) that is not viable above 75 degrees C because its adk gene has been replaced by a Geobacillus stearothermophilus ortholog revealed that growth could be restored at 78 degrees C by a vector that coexpresses polypeptides corresponding to residues 1-79 and 80-220 of AK(Tn). In contrast, PQN1 growth was not complemented by AK(Tn) fragments harboring a C156A mutation within the zinc-binding tetracysteine motif unless these fragments were fused to Thermotoga maritima chemotaxis proteins that heterodimerize (CheA and CheY) or homodimerize (CheX). This enhanced complementation is interpreted as arising from chemotaxis protein-protein interactions, since AK(Tn)-C156A fragments having only one polypeptide fused to a chemotaxis protein did not complement PQN1 to the same extent. This selection increases the maximum temperature where a PCA can be used to engineer thermostable protein complexes and to map protein-protein interactions.
Nguyen
Peter Q
PQ
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA.
Silberg
Jonathan J
JJ
D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D001411:Geobacillus stearothermophilus / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D055550:Protein Stability; D013696:Temperature; D020124:Thermotoga maritima / Q000235:genetics; D046949:Thermotoga neapolitana / Q000235:genetics / Q000378:metabolism; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010446:Peptide Fragments; D000263:Adenylate Kinase