High-Throughput Approaches for Investigating Phase Separation and Maturation of Intrinsically Disordered Proteins

Biologists tend to think of proteins in terms familiar to the macroscopic world. We speak of enzymes as "machines" that move through a defined set of motions to catalyze the chemistry of life. Similarly, we think of structural proteins like cytoskeletal filaments as the "scaffolding" that defines the size and shape of cells. However, it is increasingly apparent that a large fraction of proteins function without adopting structures that fit within the confines of these familiar analogies. These intrinsically disordered proteins (IDPs), as well as intrinsically disordered regions (IDRs) within larger proteins, are defined by a lack of stable secondary or tertiary structure, instead rapidly converting between multiple roughly isoenergetic states. A major current direction in protein science is to map out the landscape of IDR functions in mechanistic detail.

Work from many labs, including the Rosen Lab, has shown that IDRs play a critical role in phase separation, a process where molecules partition into separate compartments with different concentrations, in order to minimize the free energy of the system. Several phase-separating IDRs have been studied in detail, providing insights into the sequence and structural motifs required for phase separation. Some phase-separated IDRs also undergo a time-dependent maturation process, starting liquid-like and eventually solidifying. This process is thought to be relevant to human health, particularly in the case of the phase-separating proteins FUS and hnRNPA1, where disease-associated mutations in their IDRs accelerate solidification. Unfortunately, the experiments required to delineate the phase separation and solidification properties of IDRs are typically laborious and low-throughput. Our understanding of IDR-mediated phase separation thus remains rather piecemeal, when a more global theory would be extremely useful.

My project is to develop high-throughput, droplet microfluidics-based methods for defining fundamental sequence-function relationships in IDRs. I aim to answer these questions:

• Which features of IDR primary sequence govern the propensity to phase separate?
• Are different IDR features important in proteins that phase separate via both IDR interactions and interactions between folded domains?
• Which features of IDR primary sequences control the rate and mechanism by which phase-separated condensates solidify?

 This work will provide an empirical foundation upon which to build detailed theoretical descriptions of IDR-mediated phase separation, while yielding insights into processes that are central to large-scale organization of cells and are relevant to human health.