Luminescence complementation assay for measurement of binding to protein C-termini in live cells.
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ABSTRACT: Protein-protein interactions (PPIs) involving the extreme C-terminus serve important scaffolding and regulatory functions. Here, we leveraged NanoBiT technology to build a luminescent complementation assay for use in studying this subcategory of PPI. As a model system, we fused one component of NanoBiT to the disordered C-terminus of heat shock protein (Hsp70) and the other to its binding partner, the tetratricopeptide repeat (TPR) domain of CHIP/STUB1. We found that HEK293 cells that stably express these chimeras under a doxycycline promoter produced a robust luminescence signal. This signal was sensitive to mutations and it was further tuned by the expression of competitive C-termini. Using this system, we identified a promising, membrane permeable inhibitor of the Hsp70-CHIP interaction. More broadly, we anticipate that NanoBiT is well-suited for studying PPIs that involve C-termini.
SUBMITTER: Nadel CM
PROVIDER: S-EPMC8054679 | biostudies-literature |
REPOSITORIES: biostudies-literature
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