Genetically encoded tags for real time dissection of protein assembly in living cells

Abstract

Simple methods with straightforward readouts that enable real-time interrogation of protein quaternary structure are much needed to facilitate the physicochemical characterization of proteins at the single-cell level. After screening over a series of microtubule (MT) binders, we report herein the development of two genetically encoded tags (designated as "MoTags" for the monomer/oligomer detection tag) that can be conveniently fused to a given protein to probe its oligomeric state in cellulo when combined with routine fluorescence microscopy. In their monomeric form, MoTags are evenly distributed in the cytosol; whereas oligomerization enables MoTags to label MT or track MT tips in an oligomeric state-dependent manner. We demonstrate here the broad utility of engineered MoTags to aid the determination of protein oligomeric states, dissection of protein structure and function, and monitoring of protein-target interactions under physiological conditions in living cells.