Intracellular compartmentalization plays a pivotal role in cellular function, with membrane-bound organelles and membrane-less biomolecular 'condensates' playing key roles. These condensates, formed through liquid-liquid phase separation (LLPS), enable selective compartmentalization without the barrier of a lipid bilayer, thereby facilitating rapid formation/dissolution in response to stimuli. Intrinsically disordered proteins (IDPs) and/or proteins with intrinsically disordered regions (IDRs), which are often rich in charged and polar amino acid sequences, scaffold many condensates, often in conjunction with RNA. Comprehending the impact of IDP/IDR sequences on phase separation poses a challenge due to the extensive chemical diversity resulting from the myriad amino acids and post-translational modifications. To tackle this hurdle, one approach has been to investigate LLPS in simplified polypeptide systems, which offer a narrower scope within the chemical space for exploration. This strategy is supported by studies that have demonstrated how IDP function can largely be understood based on general chemical features, such as clusters or patterns of charged amino acids, rather than residue-level effects, and the ways in which these kinds of motifs give rise to an ensemble of conformations. Our lab has utilized complex coacervates assembled from oppositely-charged polypeptides as a simplified material analogue to the complexity of liquid-liquid phase separated biological condensates. Complex coacervation is an associative LLPS that occurs due to the electrostatic complexation of oppositely-charged macro-ions. This process is believed to be driven by the entropic gains resulting from the release of bound counterions and the reorganization of water upon complex formation. Apart from their direct applicability to IDPs, polypeptides also serve as excellent model polymers for investigating molecular interactions due to the wide range of available side-chain functionalities and the capacity to finely regulate their sequence, thus enabling precise control over interactions with guest molecules. Here, we discuss fundamental studies examining how charge patterning, hydrophobicity, chirality, and architecture affect the phase separation of polypeptide-based complex coacervates. These efforts have leveraged a combination of experimental and computational approaches that provide insight into the molecular level interactions. We also examine how these parameters affect the ability of complex coacervates to incorporate globular proteins and viruses. These efforts couple directly with our fundamental studies into coacervate formation, as such ‘guest’ molecules should not be considered as experiencing simple encapsulation and are instead active participants in the electrostatic assembly of coacervate materials. Interestingly, we observed trends in the incorporation of proteins and viruses into coacervates formed using different chain length polypeptides that are not well explained by simple electrostatic arguments and may be the result of more complex interactions between globular and polymeric species. Additionally, we describe experimental evidence supporting the potential for complex coacervates to improve the thermal stability of embedded biomolecules such as viral vaccines. Ultimately, peptide-based coacervates have the potential to help unravel the physics behind biological condensates while paving the way for innovative methods in compartmentalization, purification, and biomolecule stabilization. These advancements could have implications spanning from medicine to biocatalysis.
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Spontaneous driving forces give rise to protein−RNA condensates with coexisting phases and complex material properties
Phase separation of multivalent protein and RNA molecules underlies the biogenesis of biomolecular condensates such as membraneless organelles. In vivo, these condensates encompass hundreds of distinct types of molecules that typically organize into multilayered structures supporting the differential partitioning of molecules into distinct regions with distinct material properties. The interplay between driven (active) versus spontaneous (passive) processes that are required for enabling the formation of condensates with coexisting layers of distinct material properties remains unclear. Here, we deploy systematic experiments and simulations based on coarse-grained models to show that the collective interactions among the simplest, biologically relevant proteins and archetypal RNA molecules are sufficient for driving the spontaneous emergence of multilayered condensates with distinct material properties. These studies yield a set of rules regarding homotypic and heterotypic interactions that are likely to be relevant for understanding the interplay between active and passive processes that control the formation of functional biomolecular condensates.
more » « less- NSF-PAR ID:
- 10091547
- Publisher / Repository:
- Proceedings of the National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 116
- Issue:
- 16
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- p. 7889-7898
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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E. coli and observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active. -
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Abstract Background Biomolecular condensates are non-stoichiometric assemblies that are characterized by their capacity to spatially concentrate biomolecules and play a key role in cellular organization. Proteins that drive the formation of biomolecular condensates frequently contain oligomerization domains and intrinsically disordered regions (IDRs), both of which can contribute multivalent interactions that drive higher-order assembly. Our understanding of the relative and temporal contribution of oligomerization domains and IDRs to the material properties of in vivo biomolecular condensates is limited. Similarly, the spatial and temporal dependence of protein oligomeric state inside condensates has been largely unexplored in vivo.
Methods In this study, we combined quantitative microscopy with number and brightness analysis to investigate the aging, material properties, and protein oligomeric state of biomolecular condensates in vivo. Our work is focused on condensates formed by AUXIN RESPONSE FACTOR 19 (ARF19), a transcription factor integral to the auxin signaling pathway in plants. ARF19 contains a large central glutamine-rich IDR and a C-terminal Phox Bem1 (PB1) oligomerization domain and forms cytoplasmic condensates.
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Conclusions IDR amino acid composition affects condensate morphology and material properties. In ARF condensates, altering the amino acid composition of the IDR did not greatly affect the oligomeric state of proteins within the condensate.
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