Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate

Seim, Ian; Posey, Ammon E.; Snead, Wilton T.; Stormo, Benjamin M; Klotsa, Daphne; Pappu, Rohit V.; Gladfelter, Amy S.

doi:10.1073/pnas.2120799119

Cited by 75 publications

(67 citation statements)

References 79 publications

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“…( 73 ) discovered that the interplay between specific homotypic and heterotypic interactions of the fungal protein Whi3 influences the distribution of protein cluster sizes that forms in dilute phases that coexist with Whi3-RNA granules. Mutations to the specific interactions that generate clusters through homotypic interactions can either enhance or diminish clustering, and this has a concomitant effect on the concentrations of Whi3 proteins in the dense phase ( 73 ). Both reports highlight the importance of dilute phase clusters that are either modulated by heterotypic interactions or influence phase behavior driven by heterotypic interactions.…”

Section: Discussionmentioning

confidence: 99%

Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Kar

Hyman

Pappu

2022

Proc. Natl. Acad. Sci. U.S.A.

193

122

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Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.

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Section: Discussionmentioning

confidence: 99%

Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Kar

Hyman

Pappu

2022

Proc. Natl. Acad. Sci. U.S.A.

193

122

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“…CD measurements were carried out using a Jasco 810 spectropolarimeter scanning from 260 nm to 190 nm, with a data pitch of 1 nm and a bandwidth of 1 nm. Four to six accumulations were averaged for each spectrum with a scanning speed of 50 nm/min and a 2-s response time (106). Additional details are described in SI Appendix.…”

Section: Uv-cd Measurementsmentioning

confidence: 99%

“…Buffer MES (50 mM MES, 50 mM KCl, 2.5 mM MgCl 2 , 1 mM EGTA, pH 6.5) filtered with a 0.2 μm PES filter was used in all measurements at 30 °C. CD spectra were converted from machine units (mdeg) to mean residue ellipticity ([θ], deg cm 2 dmol −1 residue −1 ) as described (1). Peptide concentrations were determined by absorbance using an Implen NP80 nanophotometer.…”

Section: Supporting Information Textmentioning

confidence: 99%

“…CD data sets were converted to units of mean residue ellipticity as described (1) and submitted to the Dichroweb server (2) (http://dichroweb.cryst.bbk.ac.uk) for deconvolution using the CDSSTR algorithm (3) and basis sets 4, 7, and SP175t, which are optimized for the wavelength rage of 190-240 nm (4, 5). We present the α-helical, β-strand, and β-turn categories each as a sum of two sub-categories that are given in the output of the analysis.…”

Section: Supporting Information Textmentioning

confidence: 99%

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Connecting sequence features within the disordered C-terminal linker ofB. subtilisFtsZ to functions and bacterial cell division

Shinn

Cohan²,

Bullock

et al. 2022

Preprint

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Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example of such a system is the protein FtsZ that controls cell division in bacteria. FtsZs include a folded core and a C-terminal tail (CTT). The latter encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTL plus CT17 in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. To test how CTL-mediated CT17-core contacts modulate subunit interactions and GTP hydrolysis, we designed Bs-FtsZ variants where we altered the patterning of oppositely charged residues within the CTL and assessed the effects on FtsZ assembly and function. Increasing the linear segregation of oppositely charged residues within the CTL disrupts core-CTT interactions. Mutations that enhance cohesive interactions among CTTs lead to alternative, tail-driven assemblies that diminish enzymatic activity. Altering the linear segregation of oppositely charged residues within the CTL can lead to protein degradation, aberrant assembly, and disruption of cell division in vivo. The functionally relevant non-random sequence patterns identified in this work are conserved across CTLs from orthologs. Our findings highlight how sequence patterns that contribute to functions can be uncovered in IDRs, including those with minimal sequence conservation.Significance StatementConserved sequence-structure-function relationships are the defining hallmarks of well-folded proteins. These relationships can be gleaned by combining multiple sequence alignments with covariation analyses. However, the approaches that work for well-folded proteins are not transferable to intrinsically disordered regions (IDRs) because their sequences vary dramatically across orthologs. Here, we use the C-terminal autoregulatory tail (CTT) of the bacterial cell division protein FtsZ to prototype a multi-pronged approach for studying IDRs. We show how non-random sequence patterns within IDRs can be uncovered, analyzed for conservation, and redesigned to unmask their impact on molecular functions and cellular phenotypes. Through our work, we also introduce multi-pronged approaches that combine biophysical computations and biochemical experiments to understand how the CTT, as an IDR, functions.

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“…3 In fact, liquid-liquid phase separation may be more accurately described as the formation of condensate-spanning networks, due to the interplay between the propensity of the biomolecule to both phase-separate and percolate. 4,5 The arrival to prominence of membraneless organelles has sparked considerable interest in the types of molecules undergoing LLPS and the physical principles governing this process. 6 Although this type of phase transition has been reported for modular folded domains, 7 many proteins that phase separate are intrinsically disordered or have intrinsically disordered regions.…”

Section: Introductionmentioning

confidence: 99%

Phase separation in amino acid mixtures is governed by composition

Sancho

2022

Preprint

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Liquid-liquid phase separation (LLPS) has recently come to immense prominence as it is central to the formation of membraneless organelles, leading to a new paradigm of cellular organization. This type of phase transition is mediated by molecular interactions between biomolecules, including nucleic acids and both ordered and disordered proteins. In the latter case, the separation between protein-dense and dilute phases is often interpreted using models adapted from polymer theory. Specifically, the "stickers and spacers" model proposes that LLPS originates from the interplay between two classes of residues and that the main determinants for phase separation are multivalency and sequence patterning. The duality of roles of stickers (aromatics like Phe and Tyr) and spacers (Gly and polar residues) may apply more broadly in protein-like mixtures, and the presence of these two types of components alone may suffice for LLPS to take place. In order to explore this hypothesis, we use atomistic molecular dynamics simulations of capped amino-acid residues as a minimal model system. We study the behaviour of pure amino acids in water for three types of residues that are thought to play critical roles in LLPS, corresponding to the spacer and sticker categories, and their multicomponent mixtures. In agreement with previous observations, we find that the spacer type amino acids fail to phase-separate on their own, while the sticker is prone to aggregation. However, ternary amino acid mixtures involving both types of amino acids phase-separate into two phases that retain intermediate degrees of compaction and considerable fluidity. Our results suggest that LLPS is an emergent property of amino acid mixtures determined by composition.

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Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate

Cited by 75 publications

References 79 publications

Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Connecting sequence features within the disordered C-terminal linker ofB. subtilisFtsZ to functions and bacterial cell division

Phase separation in amino acid mixtures is governed by composition

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