Polymorphic protein phase transitions driven by surface anisotropy

Phase transitions of proteins are strongly influenced by surface chemical modifications or mutations. Human γD-crystallin (HGD) single-mutants have been extensively studied due to their association with juvenile cataract. However, they have also provided a rich library of molecules to examine how specific inter-protein interactions direct protein assembly, providing new insights and valuable experimental data for coarse-grained patchy-particle models. Here we demonstrate that the addition of new inter-protein interactions by mutagenesis is additive and increases the number and variety of condensed phases formed by proteins. When double mutations incorporating two specific point mutations are made, the properties of both are retained in addition to the formation a new condensed phase. We find that the HGD double-mutant P23VC110M self-assembles into spherical particles with retrograde solubility, orthorhombic crystals and needle/plate shape crystals, while retaining the ability to undergo liquid-liquid phase separation. This rich polymorphism is only partially predicted by the experimental data on the constituent single mutants. We also report a previously un-characterized amorphous protein particle, with unique properties that differ from those of protein spherulites and protein particulates previously described. The particles are amorphous, reversible with temperature, tens of microns in size and perfectly spherical. When they are grown on pristine surfaces, they appear to form by homogeneous nucleation making them unique, and possibly a new form of protein condensate. This work highlights the challenges in predicting protein behaviour, which has frustrated rational assembly and crystallization, but also provides rich data to develop new coarse-grained models to explain the observed polymorphism.