Improvement of split-luciferase complementation through stabilization of NLRP3 PYD fused luciferase fragments: in vitro and in silico studies

Split-luciferase complementary assay (SLCA) is a key method for studying protein–protein interactions. However, the NLuc and CLuc fusion proteins involved in NLRP3 PYD interactions show reduced luciferase activity due to a loss of structural stability after a freeze–thaw cycle, and homo-oligomerization and misfolding may further compromise their experimental reliability. To counteract this, reducing agents (DTT (dithiothreitol), 2-ME (2-mercaptoethanol) prevent oxidation, while glycerol stabilizes native protein conformation. Experimental results demonstrated that 10% (v/v) glycerol with 10 mM 2-ME preserves luciferase activity of NLuc-NLRP3 PYD and CLuc-NLRP3 PYD proteins after freezing and thawing. Molecular dynamics (MD) simulations results, especially in hydrogen bond analysis confirmed improved intramolecular stability and reduced solvent hydrogen interactions with proteins. These findings highlight the importance of optimizing stabilization for reliable SLCA-based protein interaction studies.