Single-chain lanthanide luminescence biosensors for cell-based imaging and screening of protein-protein interactions

Abstract

Research tools that enable imaging or analysis of protein-protein interactions (PPIs) directly within living cells provide unique and valuable biological insights and can also aid drug discovery efforts. Here, we present lanthanide-based, Förster resonance energy transfer (lanthanide-based FRET, or LRET) biosensors for time-gated luminescence (TGL) imaging or multiwell plate analysis of PPIs.Polypeptide chains comprised of an alpha helical linker flanked by a Tb(III) complex, GFP and two binding domains exhibit large differences in long-lifetime, Tb(III)-to-GFP LRET-sensitized emission between open (unbound) and closed (bound) states. We used TGL microscopy to image ca. 500% increases in Tb(III)-to-GFP LRET following rapamycin addition to NIH 3T3 cells that expressed biosensors bearing FKBP12 and the rapamycin binding domain of m-Tor (FRB) at each terminus. Much larger signal changes, up to ca. 2500%, were observed when cells were grown in 96-well or 384-well plates and analyzed using a TGL plate reader. We also measured the interaction of p53 and HDM2 and its inhibition within intact HeLa cells grown in 96-well plates and estimated a z'-factor of 0.5 for the assay. The modular design and high dynamic range of Tb(III)-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

FRET and LRET Biosensors for Cell-based Imaging and Screening of Rac1 Activation

“…Using FRET microscopy and an engineered cell line that stably expressed a Ypet/mCerulean sensor with a 20 nm ER/K linker, we observed robust Rac1 activation near protruding edges of stimulated cells. These results, along with our earlier studies, 22 demonstrate that FRET or LRET biosensors with ER/K linkers are a robust platform for engineering sensitive single cell imaging studies or higher-throughput cell-based assays of protein function in live cells.…”

“…We previously showed that biosensors which incorporate Tb(III) complexes as LRET donors, GFP as LRET acceptors and ER/K linkers can be used to craft cell-based TGL assays of protein-protein interactions that exhibit exceptional sensitivity and dynamic range. 22 Inclusion of an eDHFR domain permits self-assembly with TMP-Tb(III) complex conjugates inside live cells or in cell lysates. TGL detection of LRET offers high signal-to-background ratios that derive from the ms-scale emission decay times of Tb(III) luminescence and Tb(III)-to-GFP sensitized emission.…”

“…Rac1 activation causes PBD and GTP-bound, full-length Rac1 domains to bind one another, and the attendant conformational change repositions FRET partners to increase sensitized emission. Similar to our previously described designs, 22,23 these Rac1 biosensors incorporate two uncommon features that can enhance sensitivity and dynamic range. For one, alpha helical, ER/K linker segments separate affinity binding domains and FRET partners, maintain sensors in an extended conformation in the unbound (low activity) state, and thereby reduce baseline FRET signals.…”