Abstract
Protamines are natural polyguanidiniums, arginine(R)-rich proteins involved in the compaction of chromatin during vertebrate spermatogenesis. Salmine, a protamine isolated from salmon sperm, contains 65 mol% R residues, with positively charged guanidino (Gdm+) sidechains, and no other amino acids with ionizable or aromatic sidechains. Salmine sulfate solutions undergo liquid-liquid phase separation (LLPS) with a concentration-dependent upper critical solution temperature (UCST). The condensed liquid phase comprises 50 wt % water and >600 mg·mL−1 salmine with a constant 1:2 ratio of sulfate (SO42−) to Gdm+. Isothermal titration calorimetry, titrating Na2SO4 into salmine chloride above and below the UCST, allowed isolation of exothermic sulfate binding to salmine chloride from subsequent endothermic condensation and exothermic phase separation events. Synthetic random polyacrylate analogs of salmine, with 3-guanidinopropyl sidechains, displayed similar counterion dependent phase behavior, demonstrating that the LLPS of polyguanidiniums does not depend upon subunit sequence or polymer backbone chirality, and was due entirely to Gdm+ sidechain interactions. The results provide experimental evidence for like-charge pairing of Gdm+ sidechains, and an experimental approach for further characterizing these interactions.
Highlights
A type of liquid-liquid phase separation (LLPS), occurs when attractive interactions between solvated macromolecules are strong enough to condense the macromolecules into a separate phase, yet are weak and dynamic enough that the condensed phase forms a fluid network, rather than a solid precipitate or ionic hydrogel [1]
The results provide empirical in vitro evidence of like-charge pairing between Gdm+ groups and highlight another potential mechanism of cellular LLPS
The sensitivity to environmental conditions of the salmine and synthetic poly(Gdm+ ) condensation and phase transitions provides an experimental approach for additional study of anion effects, thermodynamics, and the structure of like-charge Gdm+ ion pairing
Summary
A type of liquid-liquid phase separation (LLPS), occurs when attractive interactions between solvated macromolecules are strong enough to condense the macromolecules into a separate phase, yet are weak and dynamic enough that the condensed phase forms a fluid network, rather than a solid precipitate or ionic hydrogel [1]. All types of non-covalent interactions between macromolecules, including ion pairing, H-bonding, π–π, π–cation, and metal-ligand interactions, can promote LLPS by complex coacervation [2,3,4]. Cellular LLPS creates transient, phase-defined cytoplasmic compartments, called membraneless organelles, in response to external stimuli [5,6,7,8,9]. Disruption of normal cellular LLPS, or gain-of-function mutations that cause abnormal cellular
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