Ionic polypeptide tags for protein phase separation

Kapelner, Rachel; Obermeyer, Allie C.

doi:10.1039/c8sc04253e

Cited by 67 publications

(98 citation statements)

References 53 publications

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“…(10) This approach has been applied to a variety of systems using both ITC and fluorescence-based binding assays to provide information on the details of charge-driven binding, 280,282 with experimental evidence correlating well with results from simulations. 283 While these binding studies were performed under very dilute conditions to avoid multi-body interactions, a number of studies have reported on both bulk complex coacervation 2,[12][13][14][15]76,[284][285][286][287][288][289][290][291] and microphase separation 73,[292][293][294][295][296][297][298][299][300][301] involving globular proteins and polyelectrolytes. Of particular note from these studies is the identification of a critical charge content necessary for complex coacervation to occur between a protein and an oppositely-charged polymer.…”

Section: Protein-polymer Coacervation and Theorymentioning

confidence: 99%

Recent progress in the science of complex coacervation

2020

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Section: Protein-polymer Coacervation and Theorymentioning

confidence: 99%

Recent progress in the science of complex coacervation

2020

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“…Therefore, decoding the sequence determinants of intrinsically disordered protein (IDP) phase separation is important for understanding the biochemistry of biomolecular condensates in physiological and pathophysiological conditions. Characterizing the effects of sequence on phase behavior is also important for the field of protein-based materials 17 , wherein proteins can be designed to have desired characteristics and programable assembly 18–20 , with applications in biotechnology such as drug delivery, cell engineering, and biomimetics 21–24 .…”

Section: Introductionmentioning

confidence: 99%

Identifying Sequence Perturbations to an Intrinsically Disordered Protein that Determine Its Phase Separation Behavior

Schuster

Dignon

Tang³

et al. 2020

Preprint

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AbstractPhase 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 bio-inspired 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. By altering the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation, we designed sequences with significantly increased phase separation propensity. 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 in spite of these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance a predictive framework and identify key biophysical principles of sequence features important to phase separation.Significance StatementMembraneless organelles are assemblies of highly concentrated biomolecules that form through a liquid-liquid phase separation process. These assemblies are often enriched in intrinsically disordered proteins, which play an important role in driving phase separation. Understanding the sequence-to-phase behavior relationship of these disordered proteins is important for understanding the biochemistry of membraneless organelles, as well as for designing synthetic organelles and biomaterials. In this work, we explore a model protein, the disordered N-terminal domain of LAF-1, and highlight how three key features of the sequence control the protein’s propensity to phase separate. Combining predictive simulations with experiments, we find that phase behavior of this model IDP is dictated by the presence of a short conserved domain, charge patterning, and arginine-tyrosine interactions.

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“…In the case of PEG/dextran ATPSs, native proteins tend to partition preferentially in the more hydrophilic dextran phase, whereas denaturedp roteins have been shown to exhibit astronger affinity for the more hydrophobic PEG phase. [72] The sequestered biomolecules remainh ighly dynamic within the liquid-like droplets, althought hey can experience subdiffusive transport. [65] In comparison, the sequestration of BSA in PAA/PAH coacervate droplets provided protection against protein denaturation on exposure to nonphysiological pH, high temperatures or urea.…”

Section: Bridging the Gap With Living Cells:i Ntracellular Biomoleculmentioning

confidence: 99%

“…[18,70] Chemical modifications have recently been used to increaset he propensityo fp roteins to undergo LLPS through, for example, protein supercharging [71] or covalent attachment of ionic polypeptidetags. [72] The sequestered biomolecules remainh ighly dynamic within the liquid-like droplets, althought hey can experience subdiffusive transport. [73] How their localised up-concentration in a crowded phase affects their biochemical reactions is an area of current study.C o-localisation of partner reactants (e.g.,e nzymes and substrates) might accelerate biochemical reactions: [74] the accumulation of hammerhead ribozymes in the dextran-rich phase of aP EG/dextran ATPS, for instance, was reported to enhance RNA catalysis rates, due to ac ombination of localised RNA up-concentration and crowding effects.…”

Section: Biomolecular Partitioninga Nd Biochemical Reactions In Membrmentioning

confidence: 99%

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

2019

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Living cells have long been a source of inspiration for chemists. Their capacity of performing complex tasks relies on the spatiotemporal coordination of matter and energy fluxes. Recent years have witnessed growing interest in the bottom‐up construction of cell‐like models capable of reproducing aspects of such dynamic organisation. Liquid–liquid phase‐separation (LLPS) processes in water are increasingly recognised as representing a viable compartmentalisation strategy through which to produce dynamic synthetic cells. Herein, we highlight examples of the dynamic properties of LLPS used to assemble synthetic cells, including their biocatalytic activity, reversible condensation and dissolution, growth and division, and recent directions towards the design of higher‐order structures and behaviour.

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Ionic polypeptide tags for protein phase separation

Abstract: Short ionic polypeptide tags were demonstrated to drive complex coacervation of globular proteins at physiological conditions while maintaining protein activity.

Cited by 67 publications

References 53 publications

Recent progress in the science of complex coacervation

Recent progress in the science of complex coacervation

Identifying Sequence Perturbations to an Intrinsically Disordered Protein that Determine Its Phase Separation Behavior

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

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