Edited by Joseph M. Jez 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 reflectinbased biophotonics, opening paths for the design of new materials with tunable properties. Cephalopods such as squid and octopuses possess an optically dynamic epithelium, enabling complex camouflage and

Reflectin proteins are widely distributed in reflective structures in cephalopods, but only in Loliginid squids are they and the sub-wavelength 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, with the resulting charge-neutralization overcoming Coulombic repulsion to progressively allow condensation and concommitant assembly to form multimeric spheres of tunable size. Structural transitions of reflectins A1 and A2 were analyzed by dynamic light scattering, transmission electron microscopy, solution small angle x-ray scattering, circular dichroism, atomic force microscopy, and fluorimetry. We analyzed the assembly behavior of phospho-mimetic, deletion, and other mutants in conjunction with pH-titration as an in vitro surrogate of phosphorylation to discover a predictive relationship between the extent of neutralization of the protein's net charge density and the size of resulting multimeric protein assemblies of narrow polydispersity. Comparison of mutants shows this sensitivity to neutralization resides in the linkers and is spatially distributed along the protein.These results are consistent with the behavior of a charge-stabilized colloidal system, while imaging of large particles, and analysis of sequence composition, suggest that assembly may proceed through a transient liquid-liquid phase separated intermediate. These results offer insights into the basis of reflectinbased tunable biophotonics and open new paths for the design of new reflectin mutants with tunable properties.

Reflectin proteins are found in the reflective Bragg lamellae in skin cells of certain squids, where they tunably control color for camouflage and communication. Initially disordered in the monomeric form, the reflectins have unusual amino acid compositions and are block copolymers consisting of polyampholytic canonical repeats interspersed with cationic linkers. In vivo, phosphorylation of the cationic linkers induces condensation, secondary folding and hierarchical assembly of the reflectins with concomitant emergence of tunable iridescence. We analyzed the folding and assembly of recombinant phospho‐mimetic and deletion mutants of reflectin, using pH titration as a surrogate for in vivo phosphorylation. Results of dynamic light scattering, transmission electron microscopy, fluorescence, circular dichroism and x‐ray scattering reveal that neutralization of the cationic linkers progressively and reversibly drives the reflectins to form monodisperse spherical assemblies of reproducible size. These analyses reveal a strongly predictive relationship between net charge density and size of the assembled reflectin multimers, with the switch appearing to be equally distributed across the spatially segregated linkers. Accompanying the progressive neutralization of Coulombic repulsion of the linkers, assembly appears to be driven by entropically driven folding of the canonical repeats into amphiphilic secondary structures that are likely to enable hierarchical assembly. These results suggest a model for tunable reflectin assembly and provide insight into the behavior of this unique, initially disordered protein.Support or Funding InformationThis research was supported by the U.S. Department of Energy, U.S. Army Research Office, and Institute of Collaborative Biotechnologies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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