Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools.

Intrinsically disordered protein regions (IDRs) are ubiquitous in all proteomes and essential to cellular function. Unlike folded domains, IDRs exist in an ensemble of rapidly changing conformations. The sequence-encoded structural biases in IDR ensembles are important for function, but are difficult to resolve. Here, we reveal hidden structural preferences in IDR ensembles in vitro with two orthogonal structural methods (SAXS and FRET), and demonstrate that these structural preferences persist in cells using live cell microscopy. Importantly, we demonstrate that some IDRs have structural preferences that can adaptively respond to even mild intracellular environment changes, while other IDRs may display a remarkable structural resilience. We propose that the ability to sense and respond to changes in cellular physicochemical composition, or to resist such changes, is a sequence-dependent property of IDRs in organisms across all kingdoms of life.

No abstract

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Methods that dynamically monitor these intracellular effects are currently lacking. Here, we leveraged the environmental sensitivity and structural plasticity of intrinsically disordered regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use in environmentally-responsive molecular tools.

No abstract