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Posey, Ammon E; Holehouse, Alex S; Pappu, Rohit V
Methods in enzymology, 2018, Letnik: 611Journal Article
There is growing interest in the topic of intracellular phase transitions that lead to the formation of biologically regulated biomolecular condensates. These condensates are membraneless bodies formed by phase separation of key protein and nucleic acid molecules from the cytoplasmic or nucleoplasmic milieus. The drivers of phase separation are referred to as scaffolds whereas molecules that preferentially partition into condensates formed by scaffolds are known as clients. Recent advances have shown that it is possible to generate physical and functional facsimiles of many biomolecular condensates in vitro. This is achieved by titrating the concentration of key scaffold proteins and solution parameters such as salt concentration, pH, or temperature. The ability to reproduce phase separation in vitro allows one to compare the relationships between information encoded in the sequences of scaffold proteins and the driving forces for phase separation. Many scaffold proteins include intrinsically disordered regions whereas others are entirely disordered. Our focus is on comparative assessments of phase separation for different scaffold proteins, specifically intrinsically disordered linear multivalent proteins. We highlight the importance of coexistence curves known as binodals for quantifying phase behavior and comparing driving forces for sequence-specific phase separation. We describe the information accessible from full binodals and highlight different methods for-and challenges associated with-mapping binodals. In essence, we provide a wish list for in vitro characterization of phase separation of intrinsically disordered proteins. Fulfillment of this wish list through key advances in experiment, computation, and theory should bring us closer to being able to predict in vitro phase behavior for scaffold proteins and connect this to the functions and features of biomolecular condensates.
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