Complete Phase Diagram for Liquid-Liquid Phase Separation of Intrinsically Disordered Proteins

Abstract

A number of intrinsically disordered proteins have been shown to self-assemble via liquid-liquid phase separation into protein-rich and dilute phases. The resulting coacervates can have important biological functions, and the ability to form these assemblies is dictated by the protein's primary amino acid sequence as well as by the solution conditions. We present a complete phase diagram for the simple coacervation of a polyampholyte intrinsically disordered protein using a field-theoretic simulation approach. We show that differences in the primary amino acid sequence and in the distribution of charged amino acids along the sequence lead to differences in the phase window for coacervation, with block-charged sequences having a larger coacervation window than sequences with a random patterning of charges. The model also captures how changing solution conditions modifies the phase diagram and can serve to guide experimental studies.

Figure 1.

Five model IDP sequences of glutamate and lysine considered in this work. Sequences and nomenclature were originally introduced in ref . Also shown are the sequence charge decoration (SCD) metric of Sawle and Ghosh and the κ parameter of Das and Pappu

Figure 2.

Left column: Excess osmotic pressure evaluated from particle MD (open circle), FTS (open square), and RPA (solid line) for model sequences sv20 (A) and sv30 (B) as a function of monomer density. Simulations were performed using a Bjerrum length of l_B = 0.033b (black) and l_B = 0.33b (red). Right column: Chemical potential as a function of monomer density for sv20 (C) and sv30 (D) with Bjerrum length l_B = 0.033b (black) and l_B = 0.33b (red). FTS results are represented with open squares, and the solid line depicts the RPA expression.

Figure 3.

(A) Snapshot of the polymer density profiles from FTS for three regions of the phase diagram: (1) a dilute solution phase; (2) a two-phase region in which a dilute supernatant and dense coacervate phase coexist; and (3) a dense protein region. (B) Coexistence curves evaluated from FTS using the Gibbs ensemble (points) and computed from RPA (solid lines). The coloring scheme is the same as that in Figure 1, representing the five different chain sequences, showing a strong charge sequence dependence on the two phase window. (C) Coexistence curves showing the reduced temperature vs monomer density. The linear scale of the horizontal axis highlights the features of the dense branch of the phase diagram but conceals the dilute branch. All simulations were performed without explicit counterions present.

Figure 4.

(A) Average radius of gyration for each of the five sequences evaluated from a single-chain particle MD simulation. The horizontal axis is the SCD metric of Sawle and Ghosh. A representative chain configuration for each sequence is also shown. All single-chain simulations we performed at l_B = 0.33b without explicit counterions present. (B) Representative configuration from a single-chain particle MD simulation showing intramolecular association between oppositely charged “patches” for the diblock chain sv30. (C) Snapshot of two representative chains from a particle MD simulation of 300 chains showing early stages of aggregation due to intermolecular association between oppositely charged groups. Chain 1 is opaque and chain 2 is shaded for contrast.

Figure 5.

(A) Coexistence points evaluated from FTS using the Gibbs ensemble at fixed l_B = 0.33b with varying excluded volume strength. Simulations were performed without explicit counterions present. The coloring scheme is the same as that in Figure 1, representing four different chain sequences. (B) Coexistence points evaluated at fixed v = 0.0069b³ and l_B = 0.33b at varying salt concentration by introducing explicit salt ions as point particles into the polymer model.