Monitoring of Protein- protein interactions in cell death using split luciferase assay: a thermodynamic perspective

Thermodynamics of protein-protein interaction is a key aspect in control of signaling pathway.Cell death modalities have different hallmark including protein complex formation likeapoptosome and necrosome. We have tried to use split luciferase complementary assay to designluminescence assay based on protein- protein interactions in apoptosis and necrosis. Apaf-1 is acytosolic multi-domain protein that forms one of the key step in the apoptosis regulatorynetwork. Following of cytochrome c release from mitochondria, it binds to WD40 repeats ofApaf-1 molecule and induces oligomerization of Apaf-1. Split luciferase complementation assayused to compare apoptosome formation by native and truncated Apaf-1. This assay uses Apaf-1tagged with either N-terminal fragment or C-terminal fragment of luciferase. Apoptosomeformation is induced inside the cells which express Apaf-1 tagged with complementaryfragments of luciferase while in cell-free system, the apoptosome formation is induced inextracts of the cells, while in cell-free system, cytochrome c dependent luciferase activity wasobserved with full length Apaf-1. The truncated mutant of Apaf-1 without WD repeats bound toendogenous Apaf-1 in a different fashion compared to native form. Molecular modelling andprotein crystallization have been used to show the role of critical residues in luciferase structurefunctionand emitted color. Different luciferase emitter may be used to visualize different proteincomplexes in cell death.