Sequence-encoded interactions determine biomolecular condensate rheology and function

Biomolecular condensates are non-stoichiometric, membraneless cellular assemblies that form and dissolve in response to a wide range of cellular signals. Condensates are known to concentrate various biopolymers, including proteins and nucleic acids, through liquid-liquid phase separation (LLPS). Condensates are thought to play important roles in organizing the cell interior, and they have emergent properties and functions. Research over the past several years has begun to uncover the molecular principles underlying condensates’ assembly and material properties, which have been linked to biological functions and pathologies. Deciphering the role of amino acid residues and the associated molecular interactions that drive the thermodynamics of LLPS, and the rheology of condensates is valuable to advance a fundamental understanding of these biologically important assemblies.Although significant progress has been made in understanding the determinants of protein phase separation, much of the focus has been on interactions from specific amino acid residues like Arginine and Tyrosine – depicted as a ‘molecular grammar’ for protein phase separation – but the contributions of other residues are less well understood. We also do not fully understand how animo acid sequences encode the material properties of the condensates, and it has been a major challenge to experimentally manipulate condensate material properties in vivo and link condensate rheology to biological functions.In this research, we investigated two systems in which molecular interactions encoded in a protein sequence were modulated to understand the role of diverse interactions contributing to protein phase separation. Furthermore, in both systems, we engineered condensates to achieve distinct biophysical properties (phase behavior and viscoelasticity) by tuning these interactions. Detailed investigations into these two systems form the two main sections of this dissertation. In the first section, we generate sequence variants of an artificial repeat intrinsically disordered polypeptide (AIDP), targeting sequence features previously overlooked in the literature. We found that multiple interactions between diverse residue pairs – and not just a handful of key residues (often referred to as ‘sticker’ residues) – work in tandem to drive the phase separation and dynamics of condensates. We further observed that condensates formed from these repeat polypeptides behaved as viscous fluids, even in the absence of glycine, which is usually considered necessary to maintain condensate fluidity. We further uncovered that a wide range of viscosities can be obtained by engineering the polypeptide sequence, highlighting the sensitive dependence of rheology on amino acid sequence.In the second part of the research, we investigated how LLPS and material properties of condensates can be tuned by modulating multivalent interactions in the naturally occurring multidomain protein PGL-3. Our aim was to develop a robust and modular engineered condensate system in C. elegans touch neurons to elucidate the correlation between physical properties of the condensates and their recruitment as cargo to large extracellular vesicles called exophers, which are extruded by the touch neurons. To achieve this, we used PGL-3 as a model protein and designed new variants by using residue-specific mutations in the intrinsically disordered region (IDR) of PGL-3, as well as by rationally swapping the IDR with select AIDP sequences from section one. The application of AIDPs resulted in enhanced intermolecular interactions, which was associated with dramatic changes in phase behavior and material properties of the condensates in vitro and when expressed in vivo in C. elegans touch neurons. We further showed that the material properties of the condensates have a strong correlation with their recruitment to exophers – i.e., condensates with slower dynamics localized relatively more to exophers than soma. Our findings suggest that naturally occurring proteins can be engineered by a judicious choice of sequence perturbations to tune the interplay between inter- and intra-domain interactions. Our work also illustrates that application of AIDPs in engineering a naturally occurring protein, PGL-3, yielded a valuable tool in modulating biophysical properties of the condensates in vivo as well as in vitro, thus enabling us to probe fundamental questions in cell biology. With the increasing emphasis on understanding the relationship between protein sequence and rheology of the condensates and how that gives rise to function, we contribute two ways by which condensate rheology can be regulated along with expanding the molecular grammar of protein phase separation.