Biomolecular Phase Separation: From Molecular Driving Forces to Macroscopic Properties

Abstract

Biological phase separation is known to be important for cellular organization, which has recently been extended to a new class of biomolecules that form liquid-like droplets coexisting with the surrounding cellular or extracellular environment. These droplets are termed membraneless organelles, as they lack a dividing lipid membrane, and are formed through liquid-liquid phase separation (LLPS). Elucidating the molecular determinants of phase separation is a critical challenge for the field, as we are still at the early stages of understanding how cells may promote and regulate functions that are driven by LLPS. In this review, we discuss the role that disorder, perturbations to molecular interactions resulting from sequence, posttranslational modifications, and various regulatory stimuli play on protein LLPS, with a particular focus on insights that may be obtained from simulation and theory. We finally discuss how these molecular driving forces alter multicomponent phase separation and selectivity.

Keywords: biomolecular condensates; intrinsically disordered proteins; multicomponent phase separation; phase separation.

Figure 1

Schematic of a single-component droplet in phase coexistence with the surrounding aqueous environment. Box shows a molecular configuration of proteins stabilizing the condensed phase at the interface. Colored side chains and zoomed insets highlight the different interaction modes occurring between protein molecules.

Figure 2

Co-phase separation of two species with similar self- and cross-interactions. A) Single component phase diagrams for Component 1 and Component 2 with tie lines at three values of the control variable: both components may phase separate (green); both 1 and 2 may phase separate, but 2 is nearing its critical point (orange); and the region where only 1 is able to form a condensed phase. B) Multicomponent phase diagram of mixtures of Components 1 and 2, with control variable indicated by color. Stars indicate different experiments conducted at different relative total compositions of the two components where 1 and 2 only contain a single component, and 3–5 contain a mixture of the two. Tie lines show the resulting concentrations within the two phases.

Figure 3

Possible shapes of multicomponent phase diagrams and the relative self- and cross-interaction strengths of components the two components. A. A cooperative condensation phase transition occurs when both components have strong self-attraction as well as a mutual attraction for one another. This is characterized by phase lines running from one axis to the other at low and high concentrations, and tie lines having a positive slope in between the two phase lines. B. Scaffold-client relationship occurs when one component has strong self-interaction and can undergo LLPS while the second cannot, but has some attraction for the first component. This results in a single phase line that intercepts the scaffold concentration axis twice. Tie lines may be positively or negatively sloped indicating recruitment and exclusion respectively. C. Cross-interaction-driven phase separation occurs when neither component has strong self-interaction, butb they have a strong mutual interaction strength. The phase lines do not intercept either axis since neither pure component is able to undergo LLPS. Tie lines are always positive because both components must be present in sufficient quantities for the condensed phase to be stabilized. D. Exclusive/Crowded phase separation occurs when one component has strong self-interaction strength, while the other has no strong interaction with itself or the first component. In this case, the first component forms a condensed phase while the second component preferentially occupies the other phase, indicated by negatively-sloped tie lines.