(467c) A Colloidal Model for Equilibrium Assembly and Liquid-Liquid Phase Separation of Reflectin Protein

Reflectin is an intrinsically disordered protein (IDP) known for its unique ability to modulate the biophotonic camouflage of cephalopods based on its assembly-induced osmotic properties. Its reversible self-assembly into discrete, size-controlled clusters and liquid-liquid phase separation are known to depend sensitively on the net protein charge, making reflectin stimuli-responsive to pH, phosphorylation, and electric fields. However, despite efforts to characterize reflectin over the past twenty years, the detailed molecular mechanisms of its assembly have yet to be identified. Here, we pursue a coarse-grained molecular understanding of reflectin assembly using a combination of experiments and simulations. We hypothesize reflectin’s assembly and phase behavior can be explained from a remarkably simple “colloidal particle” model whereby IDPs effectively interact via a short-range attractive and long-range repulsive (SA-LR) pair potential. While the SA-LR model has been applied to systems of folded proteins, it has never been used to describe the interaction between IDPs. In this work, the coarse-grained SA-LR interaction is first parameterized by small angle X-ray scattering experiments on dilute reflectin solutions. The determined interaction is then used to simulate assembly of reflectin “particles” using molecular dynamics. Remarkably, despite the simplicity of this colloidal model, the simulations successfully capture many distinctive features of reflectin assembly, including pH-dependent formation of discrete-sized nanoclusters and liquid-liquid phase separation, resulting in a proposed phase diagram for reflectin. These findings provide a mechanistic understanding of the equilibrium assembly and suggest that colloidal-scale models capture key driving forces and interactions that explain thermodynamic aspects of native reflectin behavior. Furthermore, the success of this approach suggests the potential of the colloidal interpretation of a much larger range of IDPs.