Looped liquid-liquid coexistence in protein crystallization

The potential of proteins to realize enzyme crystals or act as nanomachines in dense assemblies makes them excellent candidates for the de-novo design of biological materials. In view of the notorious complexity of protein-protein interactions, simplified models of proteins treated as patchy particles offer a promising strategy to obtain insight into the mechanism of crystallization. Here we report liquid-liquid phase separation (LLPS) with a highly asymmetric coexistence region in a computational model of rubredoxin with real molecular shape. The coexistence region terminates in both an upper (UCST) and a lower (LCST) critical solution temperature, and the complex molecular shape explains the closed-loop behavior of the LLPS. A popular conceptual framework of protein crystallization predicts that nucleation is a two-step process controlled by a metastable liquid-liquid critical point. Here, LLPS occurs via the formation of ring-like nucleation precursors if and only if the proteins are capable of crystallizing. Our findings dispute the notion that the nucleation rate may be enhanced by indirectly controlling nucleation through an independent, metastable critical point. Conversely, we show that metastable LLPS is an essential feature of crystallization.