Reflectin Proteins Bind and Reorganize Synthetic Phospholipid Vesicles.

The reflectin proteins have been extensively studied for their role in reflectance in cephalopods. In the recently evolved Loliginid squids, these proteins and the structural color they regulate are dynamically tunable, enhancing their effectiveness for camouflage and communication. In these species, the reflectins are found in highest concentrations within the structurally tunable, membrane enclosed, periodically stacked lamellae of subcellular Bragg reflectors and in the intracellular vesicles of specialized skin cells known as iridocytes and leuocophores, respectively. To better understand the interactions between the reflectins and the membrane structures that encompass them, we analyzed the interactions of two purified reflectins with synthetic phospholipid membrane vesicles similar in composition to cellular membranes, using confocal fluorescence microscopy and dynamic light scattering. The purified recombinant reflectins were found to drive multivalent vesicle agglomeration in a ratio-dependent and saturable manner. Extensive proteolytic digestion terminated with PMSF of the reflectin A1-vesicle complexes triggered energetic membrane rearrangement, resulting in vesicle fusion, fission, and tubulation. This behavior contrasted markedly with that of vesicles complexed with reflectin C, from which PMSF-terminated proteolysis only released the original size vesicles. Clues to the basis for this difference, residing in significant differences between the structures of the two reflectins, led to the suggestion that specific reflectin-membrane interactions may play a role in the ontogenetic formation, long-term maintenance, and/or dynamic behavior of their biophotonically active host membrane nanostructures. Similar energetic remodeling has been associated with osmotic stress in other membrane systems, suggesting a path to reconstitution of the biophotonic system in vitro.

Calibration between trigger and color: Neutralization of a genetically encoded coulombic switch and dynamic arrest precisely tune reflectin assembly.

Reflectin proteins are widely distributed in reflective structures in cephalopods. However, only in loliginid squids are they and the subwavelength photonic structures they control dynamically tunable, driving changes in skin color for camouflage and communication. The reflectins are block copolymers with repeated canonical domains interspersed with cationic linkers. Neurotransmitter-activated signal transduction culminates in catalytic phosphorylation of the tunable reflectins' cationic linkers; the resulting charge neutralization overcomes coulombic repulsion to progressively allow condensation, folding, and assembly into multimeric spheres of tunable well-defined size and low polydispersity. Here, we used dynamic light scattering, transmission EM, CD, atomic force microscopy, and fluorimetry to analyze the structural transitions of reflectins A1 and A2. We also analyzed the assembly behavior of phosphomimetic, deletion, and other mutants in conjunction with pH titration as an in vitro surrogate of phosphorylation. Our experiments uncovered a previously unsuspected, precisely predictive relationship between the extent of neutralization of a reflectin's net charge density and the size of resulting multimeric protein assemblies of narrow polydispersity. Comparisons of mutants revealed that this sensitivity to neutralization resides in the linkers and is spatially distributed along the protein. Imaging of large particles and analysis of sequence composition suggested that assembly may proceed through a dynamically arrested liquid-liquid phase-separated intermediate. Intriguingly, it is this dynamic arrest that enables the observed fine-tuning by charge and the resulting calibration between neuronal trigger and color in the squid. These results offer insights into the basis of reflectin-based biophotonics, opening paths for the design of new materials with tunable properties.