Charge screening and hydrophobicity drive progressive assembly and liquid-liquid phase separation of reflectin protein.

Abstract

The intrinsically disordered reflectin proteins drive tunable reflectivity for dynamic camouflage and communication in the recently evolved Loliginidae family of squid. Previous work revealed that reflectin A1 forms discrete assemblies whose size is precisely predicted by protein net charge density (NCD) and charge screening by the local anion concentration. Using dynamic light scattering (DLS), Forster resonant energy transfer (FRET) and confocal microscopy, we show that these assemblies, of which 95-99% of bulk protein in solution is partitioned into, are dynamic intermediates to liquid protein-dense condensates formed by liquid-liquid phase separation (LLPS). Increasing salt concentration drives this progression by anionic screening of the cationic protein's Coulombic repulsion, and by increasing the contribution of the hydrophobic effect which tips the balance between short-range attraction and long-range repulsion (SALR) to drive protein assembly and ultimately LLPS. Measuring fluorescence recovery after photobleaching (FRAP) and droplet fusion dynamics, we demonstrate that reflectin diffusivity in condensates is tuned by protein NCD. These results illuminate the physical processes governing reflectin A1 assembly and LLPS, and demonstrate the potential for reflectin A1 condensate-based tunable biomaterials. They also compliment previous observations of liquid phase separation in the Bragg lamellae of activated iridocytes and suggest that LLPS behavior may serve a critical role in governing the tunable and reversible dehydration of the membrane-bounded Bragg lamellae and vesicles containing reflectin in biophotonically active cells.