Multivalent polymers can control phase boundary, dynamics, and organization of liquid-liquid phase separation

Abstract

Multivalent polymers are a key structural component of many biocondensates. When interacting with their cognate binding proteins, multivalent polymers such as RNA and modular proteins have been shown to influence the liquid-liquid phase separation (LLPS) boundary to both control condensate formation and to influence condensate dynamics after phase separation. Much is still unknown about the function and formation of these condensed droplets, but changes in their dynamics or phase separation are associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer’s Disease. Therefore, investigation into how the structure of multivalent polymers relates to changes in biocondensate formation and maturation is essential to understanding and treating these diseases. Here, we use a coarse-grain, Brownian Dynamics simulation with reactive binding that mimics specific interactions in order to investigate the difference between non-specific and specific multivalent binding polymers. We show that non-specific binding interactions can lead to much larger changes in droplet formation at lower protein-polymer interaction energies than their specific, valence-limited counterparts. We also demonstrate the effects of solvent conditions and polymer length on phase separation, and we present how modulating binding energy to the polymer can change the organization of a droplet in a three component system of polymer, binding protein, and solvent. Finally, we compare the effects of surface tension and polymer binding on the condensed phase dynamics, and show that both lower protein solubilities and higher attraction/affinity of the protein to the polymer result in slower droplet dynamics. This research will help to better understand experimental systems and provides additional insight into how multivalent polymers can control LLPS.

Ion Solvation-Driven Liquid–Liquid Phase Separation in Divalent Electrolytes with Miscible Organic Solvents

“…The solvation number of DME and cosolvent can be estimated from 13 C NMR spectra at varying temperatures. According to previous work by Salama et al 19 and our group, 23 bound DME (coordinating to Mg 2+ ) and free (bulk) DME exhibit distinguishable 1 H and 13 C signals, and the difference between CH 2 and CH 3 chemical shifts of DME (Δδ DME ) is a good indicator of its coordinating state. As shown in Figure S1, Δδ ( 13 C) between CH 2 and CH 3 of bound DME and free DME is 9.4 and 13.7 ppm, respectively.…”

“…As shown in Figure S1, Δδ ( 13 C) between CH 2 and CH 3 of bound DME and free DME is 9.4 and 13.7 ppm, respectively. In Figure 2, only a set of distinct signals from DME and EMC were observed in 13 C spectrum of the lower layer of 0.5 M MgTFSI 2 -3DME-EMC at 25 °C, due to the fast exchange between the bound and the free solvent. With a value of 10.7 ppm for Δδ DME , assuming that it is the weighted average between free and bound DME, the fraction of bound DME is estimated to be 70%, corresponding to 1.7 bound DME per Mg 2+ .…”

“…1 H, 19 F, and 13 C NMR. 1 H, 19 F, and 13 C NMR measurements were primarily performed on a Varian DDRS spectrometer with a 17.6T magnet using a broad-band (BBO) probe with Larmor frequencies of 748.1 MHz for 1 H, 703.8 MHz for 19 F, and 188.1 MHz for 13 C. 90°pulse widths were measured as 20 μs for 1 H, 17 μs for 19 F, and 18 μs for 13 C. Repetition of some NMR measurements was performed on a Bruker Avance Neo spectrometer with a 11.7T magnet using a broad-band (BBO) probe with Larmor frequencies of 500.2 MHz for 1 H, 470.6 MHz for 19 F, and 125.1 MHz for 13 C. For this instrument, 90°pulse widths of 8.3 μs for 1 H, 8.5 μs for 19 F, and 9.0 μs for 13 C were determined. For quantitative NMR analysis, a 30°pulse width was used with a relaxation delay of 10 s for 1 H and 19 F and 20−60 s for 13 C to ensure full equilibration between scans.…”

“…For quantitative NMR analysis, a 30°pulse width was used with a relaxation delay of 10 s for 1 H and 19 F and 20−60 s for 13 C to ensure full equilibration between scans. NMR tubes (3 mm) were used to mitigate 1 H radiation damping, and a mixture of D 2 O (99.9 atom % D, Sigma Aldrich) with 10 mM sodium trimethylsilylpropanesulfonate (DSS, Sigma Aldrich) and DMSO-d 6 (99.5 atom % D, Sigma Aldrich) at a volume ratio of 1:2 was placed in the outer 5 mm NMR tube for signal lock and also to serve as external 1 H NMR reference (at 0 ppm with DSS and 13 C NMR reference at 39.5 ppm with DMSO). The secondary purpose of employing this mixture for the outer tube was to prevent H 2 O or DMSO from freezing when the sample temperature was lowered to −25 °C.…”

Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences

“…Unlike typical interactions in a protein complex among folded proteins, disordered polypeptides can self-assemble via multiple, relatively weak interactions between amino acids in the protein chain . Because individual interactions are weak, valency is a fundamental strategy of increasing the propensity for LLPS. − If a short peptide sequence is repeated in a larger multimer, the longer polymer has a higher probability of homotypic and heterotypic interactions, thus lowering the required free energy for phase separation. ,− In short, the multivalency of side chain interactions profoundly effects the saturation concentration ( C sat ) for protein condensation, the liquid versus gel-like properties of condensates, and the kinetics of coarsening. − The extent of multivalency can be hard coded in the amino acid sequence or through post-translational modifications. ,,− Both strategies have been explored to fine tune the extent of protein condensation under physiological conditions. ,,,− In cells, the viscoelastic properties of condensates can be altered by the presence of other multivalent polymers such as RNA. , For example, the C. elegans P granule protein LAF-1 is capable of LLPS independent of RNA in vitro, but the presence of RNA helps fluidize LAF-1 condensates .…”