Reflectin A1 is an intrinsically disordered protein (IDP) known for its ability to modulate the biophotonic camouflage of cephalopods through its reversible self-assembly into discrete, size-controlled clusters and condensed droplets. Its self-assembly are known to depend sensitively on electrostatic repulsion, making reflectin stimuli-responsive to not only pH but also salt condition. Expanding on our previous work on the pH-dependent assembly of reflectin A1, where we assumed reflectin behave as colloidal particles with short-range attraction and long-range repulsion (SA-LR) interaction, this study explores how salt concentration modulates its equilibrium self-assembly and liquid-liquid phase separation (LLPS). Using a combination of small-angle X-ray scattering, molecular dynamics simulations, and liquid-state perturbation theory, we systematically characterize the interplay between pH, salt concentration, and underlying interaction potential on assembly and phase behavior. Here, our results reveal that pH and salt regulate reflectin assembly through distinct yet complementary mechanisms, altering the balance between short-range attraction and long-range electrostatic repulsion in fundamentally different ways. By mapping a comprehensive phase diagram in the pH-salt space, we delineate monomeric, cluster-fluid, and LLPS regimes. Furthermore, by quantifying the second virial coefficient (B₂) for attractive and repulsive interactions, we establish a general criterion for predicting phase transitions based solely on the SA-LR interaction profile. This study provides validation of the SA-LR model’s ability to capture IDP assembly across both pH and salt variations, reinforcing its applicability beyond conventional globular proteins. Our findings demonstrate how different strategies for tuning effective pair interactions—whether through charge state (pH) or ionic strength (salt)—govern phase behavior. More broadly, this work highlights the predictive power of simple colloidal models in describing the complex self-assembly of intrinsically disordered proteins, offering valuable insights into phase transition mechanisms and the rational design of stimuli-responsive biomolecular materials.
