Structural biases in disordered proteins are prevalent in the cell.

Intrinsically disordered proteins and protein regions (IDPs) are prevalent in all proteomes and are essential to cellular function. Unlike folded proteins, IDPs exist in an ensemble of dissimilar conformations. Despite this structural plasticity, intramolecular interactions create sequence-specific structural biases that determine an IDP ensemble's three-dimensional shape. Such structural biases can be key to IDP function and are often measured in vitro, but whether those biases are preserved inside the cell is unclear. Here we show that structural biases in IDP ensembles found in vitro are recapitulated inside human-derived cells. We further reveal that structural biases can change in a sequence-dependent manner due to changes in the intracellular milieu, subcellular localization, and intramolecular interactions with tethered well-folded domains. We propose that the structural sensitivity of IDP ensembles can be leveraged for biological function, can be the underlying cause of IDP-driven pathology or can be used to design disorder-based biosensors and actuators.

Sequence-ensemble-function relationships for disordered proteins in live cells.

Intrinsically disordered protein regions (IDRs) are ubiquitous across all kingdoms of life and play a variety of essential cellular roles. IDRs exist in a collection of structurally distinct conformers known as an ensemble. IDR amino acid sequence determines its ensemble, which in turn can play an important role in dictating molecular function. Yet a clear link connecting IDR sequence, its ensemble properties, and its molecular function in living cells has not been systematically established. Here, we set out to test this sequence-ensemble-function paradigm using a novel computational method (GOOSE) that enables the rational design of libraries of IDRs by systematically varying specific sequence properties. Using ensemble FRET, we measured the ensemble dimensions of a library of rationally designed IDRs in human-derived cell lines, revealing how IDR sequence influences ensemble dimensions in situ. Furthermore, we show that the interplay between sequence and ensemble can tune an IDR's ability to sense changes in cell volume - a de novomolecular function for these synthetic sequences. Our results establish biophysical rules for intracellular sequence-ensemble relationships, enable a new route for understanding how IDR sequences map to function in live cells, and set the ground for the design of synthetic IDRs with de novo function.

Sequence-ensemble-function relationships for disordered proteins in live cells.

Intrinsically disordered protein regions (IDRs) are ubiquitous across all kingdoms of life and play a variety of essential cellular roles. IDRs exist in a collection of structurally distinct conformers known as an ensemble. An IDR's amino acid sequence determines its ensemble, which in turn can play an important role in dictating molecular function. Yet a clear link connecting IDR sequence, its ensemble properties, and its molecular function in living cells has not been directly established. Here, we set out to test this sequence-ensemble-function paradigm using a novel computational method (GOOSE) that enables the rational design of libraries of IDRs by systematically varying specific sequence properties. Using ensemble FRET, we measured the ensemble dimensions of a library of rationally designed IDRs in human-derived cell lines, revealing how IDR sequence influences ensemble dimensions in situ. Furthermore, we show that the interplay between sequence and ensemble can tune an IDR's ability to sense changes in cell volume - a de novo molecular function for these synthetic sequences. Our results establish biophysical rules for intracellular sequence-ensemble relationships, enable a new route for understanding how IDR sequences map to function in live cells, and set the ground for the design of synthetic IDRs with de novo function.

Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells.

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.