Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Abstract

<jats:p>Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.</jats:p>

Sequence dependent co-phase separation of RNA-protein mixtures elucidated using molecular simulations

“…We then utilize this new CG model to study the co-phase separation LAF-1 RGG protein variants with the same sequence composition but different arrangement of charged amino acids used in our previous work (37). Consistent with the expectations from previous work (31,37), we find that the phase behavior can be significantly perturbed by changes in the protein sequence, and this change in behavior also alters co-phase separation. Most importantly, we observe a significant change in terms of the co-localization of protein and RNA molecules within the condensate.…”

Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid–Liquid Phase Separation

“…Atomistic Molecular Dynamics (MD) simulations can characterize the conformational ensembles of single proteins and protein complexes [ 56 , 57 , 58 ], pinpoint the link between chemical modifications and sequence mutations, and the modulation of protein–protein and protein-DNA interactions [ 59 , 60 , 61 , 62 ], reveal the conformational heterogeneity of IDRs within small aggregates [ 63 ], and guide the development of chemically accurate coarse-grained models for LLPS [ 64 , 65 ]. Furthermore, the predictive and explanatory power of atomistic simulations is constantly being ramped up by the collective efforts to develop even more accurate atomistic force fields for IDRs [ 66 , 67 ].…”

Unraveling molecular interactions in a phase-separating protein by atomistic simulations

“…Particularly, a few studies investigated the long-range electrostatic interactions between charged amino-acids in IDP/IDRs: polymer theory has been used to rationalize how the patterning of charged residues in the sequence affects both the conformational ensemble of isolated proteins and their phase separation properties [18][19][20] . Furthermore, several evidence indicate that amino-acids with aromatic (Phe, Tyr) or large-sized, unsaturated side-chains (Arg, Gln) are key determinants of in vivo and in vitro LLPS 10,14,17,[21][22][23][24] and short-ranged attractive forces due to π-π or cation-π interactions have been invoked to rationalize these observations 25 .…”

“…Even if extremely instructive, all these studies invariantly relied on low-resolution, phenomenological CG potentials, which inherently prevented a detailed characterization of the physical interactions driving LLPS. Conversely, the contribution of high-resolution, atomistic simulations to this field has been extremely limited 15,24,38 . This unfortunate shortcoming is mainly due to the demanding computational requirements of this approach, which make the direct simulation of LLPS unfeasible with present-day computers.…”