skip to main content

Title: Impact of macromolecular crowding on RNA/spermine complex coacervation and oligonucleotide compartmentalization
We report the effect of neutral macromolecular crowders poly(ethylene glycol) (PEG) (8 kDa) and Ficoll (70 kDa) on liquid–liquid phase separation in a polyuridylic acid (polyU)/spermine complex coacervate system. The addition of PEG decreased both the amount of spermine required for phase separation and the coacervation temperature ( T C ). We interpret these effects on phase behavior as arising due to excluded volume and preferential interactions on both the secondary structure/condensation of spermine-associated polyU molecules and on the association of soluble polyU/spermine polyelectrolyte complexes to form coacervate droplets. Examination of coacervates formed in the presence of fluorescently-labeled PEG or Ficoll crowders indicated that Ficoll is accumulated while PEG is excluded from the coacervate phase, which provides further insight into the differences in phase behavior. Crowding agents impact distribution of a biomolecular solute: partitioning of a fluorescently-labeled U15 RNA oligomer into the polyU/spermine coacervates was increased approximately two-fold by 20 wt% Ficoll 70 kDa and by more than two orders of magnitude by 20 wt% PEG 8 kDa. The volume of the coacervate phase decreased in the presence of crowder relative to a dilute buffer solution. These findings indicate that potential impacts of macromolecular crowding on phase behavior and solute partitioning should be considered in model systems for intracellular membraneless organelles.  more » « less
Award ID(s):
1715984 1244180
Author(s) / Creator(s):
; ;
Date Published:
Journal Name:
Soft Matter
Page Range / eLocation ID:
368 to 378
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    When oppositely charged polyelectrolytes mix in an aqueous solution, associative phase separation gives rise to coacervates. Experiments reveal the phase diagram for such coacervates, and determine the impact of charge density, chain length and added salt. Simulations often use hybrid MC-MD methods to produce such phase diagrams, in support of experimental observations. We propose an idealized model and a simple simulation technique to investigate coacervate phase behavior. We model coacervate systems by charged bead-spring chains and counterions with short-range repulsions, of size equal to the Bjerrum length. We determine phase behavior by equilibrating a slab of concentrated coacervate with respect to swelling into a dilute phase of counterions. At salt concentrations below the critical point, the counterion concentration in the coacervate and dilute phases are nearly the same. At high salt concentrations, we find a one-phase region. Along the phase boundary, the total concentration of beads in the coacervate phase is nearly constant, corresponding to a “Bjerrum liquid''. This result can be extended to experimental phase diagrams by assigning appropriate volumes to monomers and salts. 
    more » « less
  2. Complex coacervation is an associative, liquid–liquid phase separation that can occur in solutions of oppositely-charged macromolecular species, such as proteins, polymers, and colloids. This process results in a coacervate phase, which is a dense mix of the oppositely-charged components, and a supernatant phase, which is primarily devoid of these same species. First observed almost a century ago, coacervates have since found relevance in a wide range of applications; they are used in personal care and food products, cutting edge biotechnology, and as a motif for materials design and self-assembly. There has recently been a renaissance in our understanding of this important class of material phenomena, bringing the science of coacervation to the forefront of polymer and colloid science, biophysics, and industrial materials design. In this review, we describe the emergence of a number of these new research directions, specifically in the context of polymer–polymer complex coacervates, which are inspired by a number of key physical and chemical insights and driven by a diverse range of experimental, theoretical, and computational approaches. 
    more » « less
  3. Abstract

    Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self‐assembling bacterial microcompartment (BMC) shell proteins and liquid‐liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid‐liquid interfaces between either 1) the dextran‐rich droplets and PEG‐rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two‐phase system, or 2) the polypeptide‐rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically‐driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three‐phase system wherein coacervate droplets are contained within dextran‐rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three‐phase system by changing the polyelectrolyte charge ratio. The tens‐of‐micron scale BMC shell protein‐coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.

    more » « less
  4. Abstract

    Cell‐free metabolic engineering is an emerging and promising alternative platform for the production of fuels and chemicals. In recent years, macromolecular crowding effect, which is an important function in living cells but ignored in cell‐free systems, has been transferred to cell‐free protein synthesis (CFPS). However, inhibitory effects of crowding agents on CFPS were frequently observed, and the mechanism is unclear. In this study, free Mg2+was found to be a key factor that can regulate the macromolecular crowding effect onin vitrotranscription,in vitrotranslation, and coupled transcript/translation. Addition of crowding agents (20% of Ficoll‐70 or Ficoll‐400) enhancedin vitrotranscription at an index of free Mg2+concentration (IFMC) below 2 mM but inhibited the transcription when the IFMC was higher than 2 mM. Similarly, Ficoll‐400 enhancedin vitrotranslation and coupled transcription/translation at a lower IFMC (0.1–2 mM) and inhibited the reactions at higher IFMC (>2 mM). Based on the results, CFPS systems could be further optimized by adjusting the content of crowding agents and the IFMC. Besides, the results also indicate that macromolecular crowding effect is important for maintaining the efficiency ofin vivotranscription and translation which occur at a low intracellular IFMC (<1 mM).

    more » « less

    The effects of macromolecular crowding on the transient structure of intrinsically disordered proteins is not well‐understood. Crowding by biological molecules inside cells could modulate transient structure and alter IDP function. Volume exclusion theory and observations of structured proteins suggest that IDP transient structure would be stabilized by macromolecular crowding. Amide hydrogen exchange (HX) of IDPs in highly concentrated polymer solutions would provide valuable insights into IDP transient structure under crowded conditions. Here, we have used mass spectrometry to measure HX by a transiently helical random coil domain of the activator of thyroid and retinoid receptor (ACTR) in solutions containing 300 g L−1and 400 g L−1of Ficoll, a synthetic polysaccharide, using a recently‐developed strong cation exchange‐based cleanup method [Rusinga, et al., Anal Chem 2017;89:1275–1282]. Transiently helical regions of ACTR exchanged faster in 300 g L−1Ficoll than in dilute buffer. In contrast, one transient helix exchanged more slowly in 400 g L−1Ficoll. Nonspecific interactions destabilize ACTR helicity in 300 g L−1Ficoll because ACTR engages with the Ficoll polymer mesh. In contrast, 400 g L−1Ficoll is a semi‐dilute solution where ACTR cannot engage the Ficoll mesh. At this higher concentration, volume exclusion stabilizes ACTR helicity because ACTR is compacted in interstitial spaces between Ficoll molecules. Our results suggest that the interplay between nonspecific interactions and volume exclusion in different cellular compartments could modulate IDP function by altering the stability of IDP transient structures. Proteins 2017; 85:1468–1479. © 2017 Wiley Periodicals, Inc.

    more » « less