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

Abstract

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

Structure, self-assembly, and properties of a truncated reflectin variant

“…Within this context, unique structural proteins known as reflectins have recently attracted substantial attention because of their key roles in the fascinating color-changing capabilities of cephalopods, such as the squid shown in Fig. 1 A , and have furthermore demonstrated their utility for unconventional biophotonic and bioelectronic technologies ( 11 – 40 ). For example, in vivo, Bragg stack-like ultrastructures from reflectin-based high refractive index lamellae (membrane-enclosed platelets) are responsible for the angle-dependent narrowband reflectance (iridescence) of squid iridophores, as shown in Fig.…”

“…Given reflectins’ demonstrated significance from both fundamental biology and applications perspectives, some research effort has been devoted to resolving their three-dimensional (3D) structures ( 30 , 31 , 35 – 39 ). For example, fibers drawn from full-length Euprymna scolopes reflectin 1a and films processed from truncated E. scolopes reflectin 1a were shown to possess secondary structural elements (i.e., α-helices or β-sheets) ( 30 , 31 ).…”

“…Moreover, nanoparticles assembled from both full-length and truncated Sepia officinalis reflectin 2 variants have demonstrated signatures consistent with β-sheet or α-helical secondary structure, albeit in the presence of surfactants ( 38 ). However, such studies were made exceedingly challenging by reflectins’ atypical primary sequences enriched in aromatic and charged residues, documented extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for poorly controlled aggregation ( 12 , 14 , 15 , 30 – 32 , 34 – 39 ). Consequently, the reported efforts have all suffered from multiple drawbacks, including the need for organic solvents or denaturants, the evaluation of only polydisperse or aggregated (rather than monomeric) proteins, a lack of consensus among different experimental techniques, inadequate resolution that precluded molecular-level insight, imperfect agreement between computational predictions and experimental observations, and/or the absence of conclusive correlations between structure and optical functionality.…”

Protein and Polysaccharide-Based Fiber Materials Generated from Ionic Liquids: A Review

“…ILs, then, could potentially use ionic interactions to tune the refractive properties of reflectin for specific applications. By neutralizing the linker segments in reflectin, these linker segments are able to overcome Coulombic repulsion and re-assemble into multimeric spheres of well-defined size and dispersity [93]. The wide range of anions available in ILs could result in several different self-assembly positions for reflectin for a wide variety of applications.…”

Design and fabrication of recombinant reflectin-based Bragg reflectors: bio-design engineering and photoisomerism induced wavelength modulation

“…[1] Since their discovery, researchers have been working towards the development of dynamic, optically active biomimetic camouflage technologies which exploit the unique properties of reflectins. [8][9][10][11][12] Facilitating this, recent efforts have been directed toward the characterization of reflectins in vitro, revealing properties such as pH-dependent particle-size formation, [13] the differential roles of motifs and linker regions, [6] the role of key amino-acid residues, [14] reflectin conductive properties, [15,16] and the effect of small molecules on higher-order assembly. [17] Advances in the design, fabrication, and characterization of reflectin-based materials have revealed properties such as thickness-dependent coloration, [9][10][11]18,19] broad near-infra-red (NIR) reflectance, [11] and induced light scattering, [20,21] It has also been demonstrated that the thickness (and therefore optical properties) of these materials can be controlled both pre-fabrication, by varying parameters such as flow-coating angle and sample concentration, [18,19] and post-fabrication, via vapor-induced swelling, [18,19] applying uniaxial strain, [10] or proton conduction.…”