Triplet tumbling microscopy enables in situ quantification of protein complex assembly and dynamics

Abstract

Protein-protein interactions (PPIs) mediate diverse cellular processes but are typically characterized in vitro with recombinant proteins. Current cell-compatible approaches for measuring PPIs have limited scope and often require prior knowledge of the interacting partners. Here, we develop triplet tumbling microscopy (TTM) to reveal PPIs in cells in real time. TTM reports protein complex size from rotational diffusion ("tumbling") by leveraging infrared-triggerable emission from triplet states. These long-lived triplets overcome the size limitations of traditional fluorescence anisotropy, enabling TTM to measure species from small protein complexes to organelle-scale beads. We apply TTM to detect PPIs, quantify bound fractions, and distinguish protein complexes by size. We measure diverse interactions, including rapamycin-induced dimerization, p53 homo-oligomerization, and binding of the E3-ligase E6AP to the human papilloma virus 16 E6 protein. TTM hardware is compatible with most fluorescence microscopes, providing a versatile way to access molecular insights within cells.

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