Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

Krainer, Georg; Welsh, Timothy J.; Joseph, Jerelle A.; Espinosa, Jorge R.; Wittmann, Sina; Csilléry, Ella de; Sridhar, Akshay; Toprakcioglu, Zenon; Gudiškytė, Giedre; Czekalska, Magdalena A.; Arter, William E.; Guillén-Boixet, Jordina; Franzmann, Titus M.; Qamar, Seema; George‐Hyslop, Peter St; Hyman, Anthony; Collepardo-Guevara, Rosana; Alberti, Simon; Knowles, Tuomas P. J.

doi:10.1038/s41467-021-21181-9

Cited by 289 publications

(345 citation statements)

References 125 publications

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“…In the case of A1-LCD, we suspect that increasing ionic strength screens repulsive interactions originating from the net positive charge. As the salt concentration increases further, the enhancement of hydrophobic interactions promotes distributive interactions between aromatic residues 34 , 35 . These effects depend on the specific salt type 34 , 36 .…”

Section: Resultsmentioning

confidence: 99%

“…As the salt concentration increases further, the enhancement of hydrophobic interactions promotes distributive interactions between aromatic residues 34 , 35 . These effects depend on the specific salt type 34 , 36 . To probe the properties of the dense phase, we measured the diffusion time of A1-LCD molecules inside droplets by fluorescence correlation spectroscopy (FCS) (Fig.…”

Section: Resultsmentioning

confidence: 99%

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A multi-step nucleation process determines the kinetics of prion-like domain phase separation

et al. 2021

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Compartmentalization by liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the organization of biomolecules in space and time. Here, we combine rapid-mixing time-resolved small-angle X-ray scattering (SAXS) approaches to characterize the assembly kinetics of a prototypical prion-like domain with equilibrium techniques that characterize its phase boundaries and the size distribution of clusters prior to phase separation. We find two kinetic regimes on the micro- to millisecond timescale that are distinguished by the size distribution of clusters. At the nanoscale, small complexes are formed with low affinity. After initial unfavorable complex assembly, additional monomers are added with higher affinity. At the mesoscale, assembly resembles classical homogeneous nucleation. Careful multi-pronged characterization is required for the understanding of condensate assembly mechanisms and will promote understanding of how the kinetics of biological phase separation is encoded in biomolecules.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A multi-step nucleation process determines the kinetics of prion-like domain phase separation

et al. 2021

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“…Krainer et al also reported that non-ionic and hydrophobic interactions govern phase separation at high salt concentrations [ 74 ]. They found that in the high-salt regime, where electrostatic interactions are no longer involved and screened out, the π-π pairing has a dominant role in stabilizing complexes.…”

Section: Resultsmentioning

confidence: 99%

“…Although electrostatic interactions as long-range forces primarily govern polyelectrolyte complex formation, short-range forces such as hydrophobic and π-interactions increase the stability of complexes [ 75 , 76 ]. At high salt concentrations, electrostatic interactions such as charge-charge and cation-π interactions are screened out, while π-π interactions and hydrophobic effects are less sensitive to the screening effect of salt and drive the complex formation [ 74 ]. Sequences with a fluorine substitution on the phenylalanine aromatic ring showed less hydrogen bond formation but still formed solid structures with high stability against salt likely due to increased hydrophobicity.…”

Section: Discussionmentioning

confidence: 99%

Deciphering the Role of π-Interactions in Polyelectrolyte Complexes Using Rationally Designed Peptides

2021

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Electrostatic interactions, and specifically π-interactions play a significant role in the liquid-liquid phase separation of proteins and formation of membraneless organelles/or biological condensates. Sequence patterning of peptides allows creating protein-like structures and controlling the chemistry and interactions of the mimetic molecules. A library of oppositely charged polypeptides was designed and synthesized to investigate the role of π-interactions on phase separation and secondary structures of polyelectrolyte complexes. Phenylalanine was chosen as the π-containing residue and was used together with lysine or glutamic acid in the design of positively or negatively charged sequences. The effect of charge density and also the substitution of fluorine on the phenylalanine ring, known to disrupt π-interactions, were investigated. Characterization analysis using MALDI-TOF mass spectroscopy, H NMR, and circular dichroism (CD) confirmed the molecular structure and chiral pattern of peptide sequences. Despite an alternating sequence of chirality previously shown to promote liquid-liquid phase separation, complexes appeared as solid precipitates, suggesting strong interactions between the sequence pairs. The secondary structures of sequence pairs showed the formation of hydrogen-bonded structures with a β-sheet signal in FTIR spectroscopy. The presence of fluorine decreased hydrogen bonding due to its inhibitory effect on π-interactions. π-interactions resulted in enhanced stability of complexes against salt, and higher critical salt concentrations for complexes with more π-containing amino acids. Furthermore, UV-vis spectroscopy showed that sequences containing π-interactions and increased charge density encapsulated a small charged molecule with π-bonds with high efficiency. These findings highlight the interplay between ionic, hydrophobic, hydrogen bonding, and π-interactions in polyelectrolyte complex formation and enhance our understanding of phase separation phenomena in protein-like structures.

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“…The transition of intrinsically disordered proteins to the liquid-droplet phase can be caused both by a combination of such interactions of different physical nature and by some of their types, for example, electrostatic [ 50 ]. In this case, the contribution of one type or another of interactions to the phase separation of IDPs can vary depending on the properties of the solvent [ 13 , 51 ]. Proline-rich sequences are also characteristic of some IDPs that undergo phase separation [ 52 ].…”

Section: Resultsmentioning

confidence: 99%

The Role of Non-Specific Interactions in Canonical and ALT-Associated PML-Bodies Formation and Dynamics

Fonin

Silonov

Shpironok

et al. 2021

IJMS

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In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of “small” (area less than 1 µm2) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as “seeds” of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.

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Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

Cited by 289 publications

References 125 publications

A multi-step nucleation process determines the kinetics of prion-like domain phase separation

A multi-step nucleation process determines the kinetics of prion-like domain phase separation

Deciphering the Role of π-Interactions in Polyelectrolyte Complexes Using Rationally Designed Peptides

The Role of Non-Specific Interactions in Canonical and ALT-Associated PML-Bodies Formation and Dynamics

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