Abstract
Rationally and efficiently modifying the amino-acid sequence of proteins to control their ability to undergo liquid-liquid phase separation (LLPS) on demand is not only highly desirable, but can also help to elucidate which protein features are important for LLPS. Here, we propose a computational method that couples a genetic algorithm to a sequence-dependent coarse-grained protein model to evolve the amino-acid sequences of phase-separating intrinsically disordered protein regions (IDRs), and purposely enhance or inhibit their capacity to phase-separate. We validate the predicted critical solution temperatures of the mutated sequences with ABSINTH, a more accurate all-atom model. We apply the algorithm to the phase-separating IDRs of three naturally occurring proteins, namely FUS, hnRNPA1 and LAF1, as prototypes of regions that exist in cells and undergo homotypic LLPS driven by different types of intermolecular interaction, and we find that the evolution of amino-acid sequences towards enhanced LLPS is driven in these three cases, among other factors, by an increase in the average size of the amino acids. However, the direction of change in the molecular driving forces that enhance LLPS (such as hydrophobicity, aromaticity and charge) depends on the initial amino-acid sequence. Finally, we show that the evolution of amino-acid sequences to modulate LLPS is strongly coupled to the make-up of the medium (e.g. the presence or absence of RNA), which may have significant implications for our understanding of phase separation within the many-component mixtures of biological systems.
Highlights
Liquid–liquid phase separation (LLPS) of multivalent biomolecules is an important mechanism employed by cells to control the spatio-temporal organisation of their many components [1, 2]
We apply the algorithm to the phase-separating intrinsically disordered protein regions (IDRs) of three naturally occurring proteins, namely Fused in Sarcoma (FUS), hnRNPA1 and LAF1, as prototypes of regions that exist in cells and undergo homotypic LLPS driven by different types of intermolecular interaction, and we find that the evolution of amino-acid sequences towards enhanced LLPS is driven in these three cases, among other factors, by an increase in the average size of the amino acids
Protein condensates formed by the process of liquid–liquid phase separation (LLPS) play diverse roles inside cells—from spatio-temporal compartmentalisation to speeding up chemical reactions
Summary
Liquid–liquid phase separation (LLPS) of multivalent biomolecules (e.g. proteins and nucleic acids) is an important mechanism employed by cells to control the spatio-temporal organisation of their many components [1, 2]. Biomolecular condensates, or membraneless organelles, such as stress granules [3], P-granules [4, 5], the nephrin–NCK–WASP system [6] and the nucleoli [7], are formed by LLPS and have diverse biological functions. Some biomolecular condensates emerge spontaneously inside cells without as-yet clearly identified functions; it has been hypothesised that some of these might be implicated in the emergence of phase-separation-related pathologies [15]. Biomolecular condensates have been proposed as promising new tools to partition anti-cancer drugs preferentially to cancer cells [16]. Such a richness of behaviours highlights the importance of learning to design protein mutations that can alter the stabilities of condensates
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