Uncovering the Role of Surface Residues and Buffer Composition in Liquid-Liquid Phase Separation of Eye Lens Crystallins from an Antarctic Toothfish

Abstract

The Antarctic toothfish, Dissostichus mawsoni, lives in the Southern Ocean around Antarctica, requiring adaptations to deal with the challenges of living in a subfreezing environment. One such adaptation is that the D. mawsoni eye lens is completely resistant to liquid-liquid phase separation (LLPS) of the eye lens proteins, also known as cold cataract. Despite this observation, a sub-fraction of the D. mawsoni eye lens proteins was found to undergo LLPS above the body temperature of the toothfish. Six γ-crystallins were identified from this fraction and characterized for their LLPS behavior and general features of stability. No obvious relationship could be discerned between the critical temperatures at which these γ-crystallins undergo LLPS and the various measures of stability. Additionally, mutagenesis of predicted surface exposed residues was performed to study their role in the LLPS behavior. Mutagenesis revealed that replacing exposed arginine for lysine results in a decrease of the phase transition temperature, while replacing lysine with arginine had the opposite effect, but similar magnitude. These finding highlights the importance of arginine in increasing attractive protein-protein interactions even when the site specific charge is largely unchanged. Furthermore, we have shown that the components of the buffer solution can further modulate the phase transition temperature of these crystallins.