Liquid-liquid phase separation (LLPS) is widely utilized by the cell to organize and regulate various biochemical processes. Although the LLPS of proteins is known to occur in a sequence dependent manner, it is unclear how sequence properties dictate the nature of the phase transition and thereby influence condensed phase morphology. In this work, we have utilized grand canonical Monte Carlo simulations for a simple coarsegrained model of disordered proteins to systematically investigate how sequence distribution, sticker fraction and chain length influence the phase behavior and regulate the formation of finite-size aggregates preempting macroscopic phase separation for some sequences. We demonstrate that a normalized sequence charge decoration (SCD) parameter establishes a "soft" criterion for predicting the underlying phase transition of a model protein. Additionally, we find that this order parameter is strongly correlated to the critical density for phase separation, highlighting an unambiguous connection between sequence distribution and condensed phase density. Results obtained from an analysis of the order parameter reveals that at sufficiently long chain lengths, the vast majority of sequences are likely to phase separate. Our results predict that classical LLPS should be the dominant phase transition for disordered proteins and suggests a possible reason behind recent findings of widespread phase separation throughout living cells.

Endogenous biomolecular condensates, comprised of a multitude of proteins and RNAs, can organize into multiphasic structures, with compositionally-distinct phases. This multiphasic organization is generally understood to be critical for facilitating their proper biological function. However, the biophysical principles driving multiphase formation are not completely understood. Here, we utilize in vivo condensate reconstitution experiments and coarse-grained molecular simulations to investigate how oligomerization and sequence interactions modulate multiphase organization in biomolecular condensates. We demonstrate that increasing the oligomerization state of an intrinsically disordered protein region (IDR) results in enhanced immiscibility and multiphase formation. Interestingly, we found that oligomerization tunes the miscibility of IDRs in an asymmetric manner, with the effect being more pronounced when the IDR exhibiting stronger homotypic IDR interactions is oligomerized. Our findings suggest that oligomerization is a flexible biophysical mechanism which cells can exploit to tune the internal organization of biomolecular condensates and their associated biological functions.

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Liquid-liquid phase separation (LLPS) is widely utilized by the cell to organize and regulate various biochemical processes. Although the LLPS of proteins is known to occur in a sequence dependent manner, it is unclear how sequence properties dictate the nature of the phase transition and thereby influence condensed phase morphology. In this work, we have utilized grand canonical Monte Carlo simulations for a simple coarse-grained model of disordered proteins to systematically investigate how sequence distribution, sticker fraction and chain length influence the phase behavior and regulate the formation of finite-size aggregates preempting macroscopic phase separation for some sequences. We demonstrate that a normalized sequence charge decoration (SCD) parameter establishes a ``soft" criterion for predicting the underlying phase transition of a model protein. Additionally, we find that this order parameter is strongly correlated to the critical density for phase separation, highlighting an unambiguous connection between sequence distribution and condensed phase density. Results obtained from an analysis of the order parameter reveals that at sufficiently long chain lengths, the vast majority of sequences are likely to phase separate. Our results predict that classical LLPS should be the dominant phase transition for disordered proteins and suggests a possible reason behind recent findings of widespread phase separation throughout living cells.