Controlling Liquid–Liquid Phase Separation of Cold-Adapted Crystallin Proteins from the Antarctic Toothfish

Alteration of Microstructure in Biopolymeric Hydrogels via Compositional Modification of Resilin-Like Polypeptides

“…These observations suggest that, in the microstructured RLP hydrogels (see below), it is only the higher concentration RLP phase that comprises a cross-linked, percolated polymer network. As expected, the phase diagram (Figure A) has the typical shape of a polymer with UCST transition and is consistent with other proteins that undergo LLPS upon cooling, as is in the case of γ-crystallins , or other RLP constructs. , Interestingly, in such proteins, the LLPS transition is enhanced with increasing the content of both arginine and aromatic amino acids, as we have shown here in our atomistic simulations (Figure A). The transition temperatures shown in the coexistence curve suggest conditions suitable for cell culture applications, as the solutions phase separate close to physiological conditions and at concentrations required for effective cross-linking as detailed above.…”

Chemical Properties Determine Solubility and Stability in βγ‐Crystallins of the Eye Lens

“…[234] Although the whole lens of D. mawsoni resists cold cataract down to at least À 12°C, below which it freezes solid, [235] the individual γ- crystallins vary greatly in cold-cataract resistance despite their high sequence identity. [236] Wild-type toothfish γM8b phase separates at À 4.8°C, but mutating only three Arg residues on the surface to Lys makes it more resistant to cold cataract than any of the wild-type proteins tested, with an onset temperature of À 13.7°C, illustrating the importance of specific proteinprotein interactions. Conversely, mutating three Lys residues to Arg raises the onset temperature of LLPS by an equivalent amount, to 3.5°C, [236] thereby demonstrating that LLPS can be controlled by making subtle changes to protein surface properties, even in well-folded proteins.…”

“…The Antarctic toothfish, Dissostichus mawsoni , has at least 13 expressed γ‐crystallin paralogues [234] . Although the whole lens of D. mawsoni resists cold cataract down to at least −12 °C, below which it freezes solid, [235] the individual γ‐crystallins vary greatly in cold‐cataract resistance despite their high sequence identity [236] . Wild‐type toothfish γM8b phase separates at −4.8 °C, but mutating only three Arg residues on the surface to Lys makes it more resistant to cold cataract than any of the wild‐type proteins tested, with an onset temperature of −13.7 °C, illustrating the importance of specific protein‐protein interactions.…”

“…Wild‐type toothfish γM8b phase separates at −4.8 °C, but mutating only three Arg residues on the surface to Lys makes it more resistant to cold cataract than any of the wild‐type proteins tested, with an onset temperature of −13.7 °C, illustrating the importance of specific protein‐protein interactions. Conversely, mutating three Lys residues to Arg raises the onset temperature of LLPS by an equivalent amount, to 3.5 °C, [236] thereby demonstrating that LLPS can be controlled by making subtle changes to protein surface properties, even in well‐folded proteins. The importance of specific surface interactions were hinted at in early studies, for example the finding that phase separation induces major changes to the Raman spectrum of tyrosine in lens proteins [237] .…”

Driving Forces of Liquid–Liquid Phase Separation in Biological Systems

“…The majority of these publications are focused on the analysis of macromolecules (proteins and RNA/DNA) enriched in membrane-less organelles and their structural features [7,9,10,11,12,13,14,15,16,17], as well as on the analysis of model systems capable to isolate protein-rich phases under certain conditions in vitro as well as in vivo. Typically, the model systems used in such studies include different phase-separating proteins, such as tau-protein [18], elastin-like polypeptides [19], fused in sarcoma (FUS) [20], transactive response element (TAR) DNA-binding protein of 43 kDa (TDP-43) [21], structural γ-crystallins [22], polyQ-protein Whi3 [23], proteasomal shuttle factor UBQLN2 [24], nucleophosmin (NPM1) [25], Numb/Pon complex from Drosophila neuroblast [26], C9orf72 dipeptide repeat proteins [27], multivalent signaling proteins [28], and many others. There are several reports [29,30] indicating that the solvent properties of membrane-less organelles and protein-rich phases are different from those of the environment from which these phases originate.…”