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 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.