Proteins that undergo liquid–liquid phase separation (LLPS) have been shown to play a critical role in many physiological functions through formation of condensed liquid-like assemblies that function ...as membraneless organelles within biological systems. To understand how different proteins may contribute differently to these assemblies and their functions, it is important to understand the molecular driving forces of phase separation and characterize their phase boundaries and material properties. Experimental studies have shown that intrinsically disordered regions of these proteins are a major driving force, as many of them undergo LLPS in isolation. Previous work on polymer solution phase behavior suggests a potential correspondence between intramolecular and intermolecular interactions that can be leveraged to discover relationships between single-molecule properties and phase boundaries. Here, we take advantage of a recently developed coarse-grained framework to calculate the θ temperature Tθ
, the Boyle temperature TB
, and the critical temperature Tc
for 20 diverse protein sequences, and we show that these three properties are highly correlated. We also highlight that these correlations are not specific to our model or simulation methodology by comparing between different pairwise potentials and with data from other work. We, therefore, suggest that smaller simulations or experiments to determine Tθ
or TB
can provide useful insights into the corresponding phase behavior.
Droplet organelles? Courchaine, Edward M; Lu, Alice; Neugebauer, Karla M
The EMBO journal,
01 August 2016, Letnik:
35, Številka:
15
Journal Article
Recenzirano
Odprti dostop
Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular activities, and they have ...been variously described as bodies, granules, or organelles. Recent evidence suggests that a liquid–liquid phase separation (LLPS) process may drive their formation, possibly justifying the unifying term “droplet organelle”. A veritable deluge of recent publications points to the importance of low‐complexity proteins and RNA in determining the physical properties of phase‐separated structures. Many of the proteins linked to such structures are implicated in human diseases, such as amyotrophic lateral sclerosis (ALS). We provide an overview of the organizational principles that characterize putative “droplet organelles” in healthy and diseased cells, connecting protein biochemistry with cell physiology.
Non‐membrane‐bound cellular structures such as nucleoli, stress granules, Cajal and P bodies have been long established. Recent data reviewed by Neugebauer and colleagues delineate liquid–liquid phase separation processes that underlie the dynamic nature of these organelles composed of low‐complexity proteins and RNA.
Here we review the current understanding of molecular interactions that govern liquid–liquid phase separation (LLPS) of biological condensates. The connection between sequence, chain conformation, ...and phase separation of intrinsically disordered proteins (IDPs) and their model polyampholytes is discussed. In particular, we highlight how the charge pattern influences the conformation and phase behavior of natural IDPs. We then describe recent results from theoretical treatments of polyampholytes implementing random phase approximation, field-theoretic simulations, and transfer matrix theory that show an increase in charge segregation results in an increased tendency to phase separate.
Display omitted
Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human ...stress granules. Here, we test the possibility that the multivalent RNA‐binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) condenses with RNA via liquid–liquid phase separation (LLPS) and that N protein can be recruited in phase‐separated forms of human RNA‐binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N‐terminal IDR and central‐linker IDR, as well as the folded C‐terminal oligomerization domain, while the folded N‐terminal domain and the C‐terminal IDR are not required. N protein phase separation is induced by addition of non‐specific RNA. In addition, N partitions in vitro into phase‐separated forms of full‐length human hnRNPs (TDP‐43, FUS, hnRNPA2) and their low‐complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS‐CoV‐2 viral genome packing and in host‐protein co‐opting necessary for viral replication and infectivity.
SYNOPSIS
Viruses can assemble nucleocapsid protein (N) and genomic RNA in dynamic compartments containing host ribonucleoproteins. We show SARS‐CoV‐2 N undergoes liquid‐liquid phase separation (LLPS) with RNA and enters droplets formed by human hnRNPs.
N phase separation in vitro in physiological buffer conditions is enhanced by RNA.
N‐terminal and linker disordered domains and C‐terminal folded dimerization domain are essential for robust LLPS.
RNA sequence specificity is not necessary for enhancing N LLPS.
N partitions into phase separated forms of hnRNPA2, TDP‐43, and FUS.
The nucleocapsid protein from SARS‐CoV‐2 undergoes liquid‐liquid phase separation with RNA and can partition into droplets formed by TDP‐43, FUS or hnRNPA2.
Biologically functional liquid-liquid phase separation of intrinsically disordered proteins (IDPs) is driven by interactions encoded by their amino acid sequences. Little is currently known about the ...molecular recognition mechanisms for distributing different IDP sequences into various cellular membraneless compartments. Pertinent physics was addressed recently by applying random-phase-approximation (RPA) polymer theory to electrostatics, which is a major energetic component governing IDP phase properties. RPA accounts for charge patterns and thus has advantages over Flory-Huggins (FH) and Overbeek-Voorn mean-field theories. To make progress toward deciphering the phase behaviors of multiple IDP sequences, the RPA formulation for one IDP species plus solvent is hereby extended to treat polyampholyte solutions containing two IDP species plus solvent. The new formulation generally allows for binary coexistence of two phases, each containing a different set of volume fractions ( φ 1 , φ 2 ) for the two different IDP sequences. The asymmetry between the two predicted coexisting phases with regard to their φ 1 φ 2 ratios for the two sequences increases with increasing mismatch between their charge patterns. This finding points to a multivalent, stochastic, 'fuzzy' mode of molecular recognition that helps populate various IDP sequences differentially into separate phase compartments. An intuitive illustration of this trend is provided by FH models, whereby a hypothetical case of ternary coexistence is also explored. Augmentations of the present RPA theory with a relative permittivity ϵ r ( φ ) that depends on IDP volume fraction φ = φ 1 + φ 2 lead to higher propensities to phase separate, in line with the case with one IDP species we studied previously. Notably, the cooperative, phase-separation-enhancing effects predicted by the prescriptions for ϵ r ( φ ) we deem physically plausible are much more prominent than that entailed by common effective medium approximations based on Maxwell Garnett and Bruggeman mixing formulas. Ramifications of our findings on further theoretical development for IDP phase separation are discussed.
We present improvements to the hydropathy scale (HPS) coarse‐grained (CG) model for simulating sequence‐specific behavior of intrinsically disordered proteins (IDPs), including their liquid–liquid ...phase separation (LLPS). The previous model based on an atomistic hydropathy scale by Kapcha and Rossky (KR scale) is not able to capture some well‐known LLPS trends such as reduced phase separation propensity upon mutations (R‐to‐K and Y‐to‐F). Here, we propose to use the Urry hydropathy scale instead, which was derived from the inverse temperature transitions in a model polypeptide with guest residues X. We introduce two free parameters to shift (Δ) and scale (µ) the overall interaction strengths for the new model (HPS‐Urry) and use the experimental radius of gyration for a diverse group of IDPs to find their optimal values. Interestingly, many possible (Δ, µ) combinations can be used for typical IDPs, but the phase behavior of a low‐complexity (LC) sequence FUS is only well described by one of these models, which highlights the need for a careful validation strategy based on multiple proteins. The CG HPS‐Urry model should enable accurate simulations of protein LLPS and provide a microscopically detailed view of molecular interactions.
A guiding principle of biology is that biochemical reactions must be organized in space and time. One way this spatio-temporal organization is achieved is through liquid-liquid phase separation ...(LLPS), which generates biomolecular condensates. These condensates are dynamic and reactive, and often contain a complex mixture of proteins and nucleic acids. In this review, we discuss how underlying physical and chemical processes generate internal condensate architectures. We then outline the diverse condensate architectures that are observed in biological systems. Finally, we discuss how specific condensate organization is critical for specific biological functions.
To date, the recently discovered SARS‐CoV‐2 virus has afflicted >6.9 million people worldwide and disrupted the global economy. Development of effective vaccines or treatments for SARS‐CoV‐2 ...infection will be aided by a molecular‐level understanding of SARS‐CoV‐2 proteins and their interactions with host cell proteins. The SARS‐CoV‐2 nucleocapsid (N) protein is highly homologous to the N protein of SARS‐CoV, which is essential for viral RNA replication and packaging into new virions. Emerging models indicate that nucleocapsid proteins of other viruses can form biomolecular condensates to spatiotemporally regulate N protein localization and function. Our bioinformatic analyses, in combination with pre‐existing experimental evidence, suggest that the SARS‐CoV‐2 N protein is capable of forming or regulating biomolecular condensates in vivo by interaction with RNA and key host cell proteins. We discuss multiple models, whereby the N protein of SARS‐CoV‐2 may harness this activity to regulate viral life cycle and host cell response to viral infection.
PDF
