A colloidal model for the equilibrium assembly and liquid-liquid phase separation of the reflectin A1 protein.

Reflectin is an intrinsically disordered protein (IDP) known for its ability to modulate the biophotonic camouflage of cephalopods based on its assembly-induced osmotic properties. Its reversible self-assembly into discrete, size-controlled clusters and condensed droplets are known to depend sensitively on the net protein charge, making reflectin stimuli-responsive to pH, phosphorylation, and electric fields. Despite considerable efforts to characterize this behavior, the detailed physical mechanisms of reflectin's assembly are yet fully understood. Here, we pursue a coarse-grained molecular understanding of reflectin assembly using a combination of experiments and simulations. We hypothesize that reflectin assembly and phase behavior can be explained from a remarkably simple colloidal model whereby individual protein monomers effectively interact via a short-range attractive and long-range repulsive (SA-LR) pair potential. We parameterize a coarse-grained SA-LR interaction potential for reflectin A1 from small angle X-ray scattering measurements, and then extend it to a range of pH using Gouy-Chapman theory to model monomer-monomer electrostatic interactions. The pH-dependent SA-LR interaction is then used in molecular dynamics simulations of reflectin assembly, which successfully capture a number of qualitative features of reflectin, including pH-dependent formation of discrete-sized nanoclusters and liquid-liquid phase separation at high pH, resulting in a putative phase diagram for reflectin. Importantly, we find that at low pH, size-controlled reflectin clusters are equilibrium assemblies, which dynamically exchange protein monomers to maintain an equilibrium size distribution. These findings provide a mechanistic understanding of the equilibrium assembly of reflectin, and suggest that colloidal-scale models capture key driving forces and interactions to explain thermodynamic aspects of native reflectin behavior. Furthermore, the success of SA-LR interactions presented in this study demonstrates the potential of a colloidal interpretation of interactions and phenomena in a range of IDPs.

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

Naturally occurring and recombinant protein-based materials are frequently employed for the study of fundamental biological processes and are often leveraged for applications in areas as diverse as electronics, optics, bioengineering, medicine, and even fashion. Within this context, unique structural proteins known as reflectins have recently attracted substantial attention due to their key roles in the fascinating color-changing capabilities of cephalopods and their technological potential as biophotonic and bioelectronic materials. However, progress toward understanding reflectins has been hindered by their atypical aromatic and charged residue-enriched sequences, extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for aggregation. Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a straightforward mechanical agitation-based methodology for controlling this variant's hierarchical assembly, and establish a direct correlation between the protein's structural characteristics and intrinsic optical properties. Altogether, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells' color-changing functionalities, and may inform new research directions across biochemistry, cellular biology, bioengineering, and optics.

A Mini-Review on Reflectins, from Biochemical Properties to Bio-Inspired Applications.

Some cephalopods (squids, octopuses, and cuttlefishes) produce dynamic structural colors, for camouflage or communication. The key to this remarkable capability is one group of specialized cells called iridocytes, which contain aligned membrane-enclosed platelets of high-reflective reflectins and work as intracellular Bragg reflectors. These reflectins have unusual amino acid compositions and sequential properties, which endows them with functional characteristics: an extremely high reflective index among natural proteins and the ability to answer various environmental stimuli. Based on their unique material composition and responsive self-organization properties, the material community has developed an impressive array of reflectin- or iridocyte-inspired optical systems with distinct tunable reflectance according to a series of internal and external factors. More recently, scientists have made creative attempts to engineer mammalian cells to explore the function potentials of reflectin proteins as well as their working mechanism in the cellular environment. Progress in wide scientific areas (biophysics, genomics, gene editing, etc.) brings in new opportunities to better understand reflectins and new approaches to fully utilize them. The work introduced the composition features, biochemical properties, the latest developments, future considerations of reflectins, and their inspiration applications to give newcomers a comprehensive understanding and mutually exchanged knowledge from different communities (e.g., biology and material).

Natural biopolymers as proton conductors in bioelectronics.

Bioelectronic devices sense or deliver information at the interface between living systems and electronics by converting biological signals into electronic signals and vice-versa. Biological signals are typically carried by ions and small molecules. As such, ion conducting materials are ideal candidates in bioelectronics for an optimal interface. Among these materials, ion conducting polymers that are able to uptake water are particularly interesting because, in addition to ionic conductivity, their mechanical properties can closely match the ones of living tissue. In this review, we focus on a specific subset of ion-conducting polymers: proton (H+ ) conductors that are naturally derived. We first provide a brief introduction of the proton conduction mechanism, and then outline the chemical structure and properties of representative proton-conducting natural biopolymers: polysaccharides (chitosan and glycosaminoglycans), peptides and proteins, and melanin. We then highlight examples of using these biopolymers in bioelectronic devices. We conclude with current challenges and future prospects for broader use of natural biopolymers as proton conductors in bioelectronics and potential translational applications.

Structures, Organization, and Function of Reflectin Proteins in Dynamically Tunable Reflective Cells.

The reversible assembly of reflectin proteins drives dynamic iridescence in cephalopods. Squid dynamically tune the intensity and colors of iridescence generated by constructive interference from intracellular Bragg reflectors in specialized skin cells called iridocytes. Analysis of the tissue specificity of reflectin subtypes reveals that tunability is correlated with the presence of one specific reflectin sequence. Differential phosphorylation and dephosphorylation of the reflectins in response to activation by acetylcholine, as well as differences in their tissue-specific and subcellular spatial distributions, further support the suggestion of different roles for the different reflectin subtypes.

Voltage-calibrated, finely tunable protein assembly.

Neuronally triggered phosphorylation drives the calibrated and cyclable assembly of the reflectin signal transducing proteins, resulting in their fine tuning of colours reflected from specialized skin cells in squid for camouflage and communication. In close parallel to this physiological behaviour, we demonstrate for the first time that electrochemical reduction of reflectin A1, used as a surrogate for charge neutralization by phosphorylation, triggers voltage-calibrated, proportional and cyclable control of the size of the protein's assembly. Electrochemically triggered condensation, folding and assembly were simultaneously analysed using in situ dynamic light scattering, circular dichroism and UV absorbance spectroscopies. The correlation of assembly size with applied potential is probably linked to reflectin's mechanism of dynamic arrest, which is controlled by the extent of neuronally triggered charge neutralization and the corresponding fine tuning of colour in the biological system. This work opens a new perspective on electrically controlling and simultaneously observing reflectin assembly and, more broadly, provides access to manipulate, observe and electrokinetically control the formation of intermediates and conformational dynamics of macromolecular systems.

Cephalopod-Mimetic Tunable Photonic Coatings Assembled from Quasi-Monodispersed Reflectin Protein Nanoparticles.

The remarkable dynamic camouflage ability of cephalopods arises from precisely orchestrated structural changes within their chromatophores and iridophores photonic cells. This mesmerizing color display remains unmatched in synthetic coatings and is regulated by swelling/deswelling of reflectin protein nanoparticles, which alters platelet dimensions in iridophores to control photonic patterns according to Bragg's law. Toward mimicking the photonic response of squid's skin, reflectin proteins from Sepioteuthis lessioniana were sequenced, recombinantly expressed, and self-assembled into spherical nanoparticles by conjugating reflectin B1 with a click chemistry ligand. These quasi-monodisperse nanoparticles can be tuned to any desired size in the 170-1000 nm range. Using Langmuir-Schaefer and drop-cast deposition methods, ligand-conjugated reflectin B1 nanoparticles were immobilized onto azide-functionalized substrates via click chemistry to produce monolayer amorphous photonic structures with tunable structural colors based on average particle size, paving the way for the fabrication of eco-friendly, bioinspired color-changing coatings that mimic cephalopods' dynamic camouflage.

Long-Range Proton Transport in Films from a Reflectin-Derived Polypeptide.

Protein- and peptide-based proton conductors have been extensively studied because of their important roles in biological processes and established potential for bioelectronic device applications. However, despite much progress, the demonstration of long-range proton transport for such materials has remained relatively rare. Herein, we fabricate, electrically interrogate, and physically characterize films from a reflectin-derived polypeptide. The electrical measurements indicate that device-integrated films exhibit proton conductivities with values of ∼0.4 mS/cm and sustain proton transport over distances of ∼1 mm. The accompanying physical characterization indicates that the polypeptide possesses characteristics analogous to those of the parent protein class and furnishes insight into the relationship between the polypeptide's electrical functionality and structure in the solid state. When considered together, our findings hold significance for the continued development and engineering of not only reflectin-based materials but also other bioinspired proton conductors.

Electrochemistry as a surrogate for protein phosphorylation: voltage-controlled assembly of reflectin A1.

Phosphorylation is among the most widely distributed mechanisms regulating the tunable structure and function of proteins in response to neuronal, hormonal and environmental signals. We demonstrate here that the low-voltage electrochemical reduction of histidine residues in reflectin A1, a protein that mediates the neuronal fine-tuning of colour reflected from skin cells for camouflage and communication in squids, acts as an in vitro surrogate for phosphorylation in vivo, driving the assembly previously shown to regulate its function. Using micro-drop voltammetry and a newly designed electrochemical cell integrated with an instrument measuring dynamic light scattering, we demonstrate selective reduction of the imidazolium side chains of histidine in monomers, oligopeptides and this complex protein in solution. The formal reduction potential of imidazolium proves readily distinguishable from those of hydronium and primary amines, allowing unequivocal confirmation of the direct and energetically selective deprotonation of histidine in the protein. The resulting 'electro-assembly' provides a new approach to probe, understand, and control the mechanisms that dynamically tune protein structure and function in normal physiology and disease. With its abilities to serve as a surrogate for phosphorylation and other mechanisms of charge neutralization, and to potentially isolate early intermediates in protein assembly, this method may be useful for analysing never-before-seen early intermediates in the phosphorylation-driven assembly of other proteins in normal physiology and disease.

Origin of the Reflectin Gene and Hierarchical Assembly of Its Protein.

Cephalopods, the group of animals including octopus, squid, and cuttlefish, have remarkable ability to instantly modulate body coloration and patterns so as to blend into surrounding environments [1, 2] or send warning signals to other animals [3]. Reflectin is expressed exclusively in cephalopods, filling the lamellae of intracellular Bragg reflectors that exhibit dynamic iridescence and structural color change [4]. Here, we trace the possible origin of the reflectin gene back to a transposon from the symbiotic bioluminescent bacterium Vibrio fischeri and report the hierarchical structural architecture of reflectin protein. Intrinsic self-assembly, and higher-order assembly tightly modulated by aromatic compounds, provide insights into the formation of multilayer reflectors in iridophores and spherical microparticles in leucophores and may form the basis of structural color change in cephalopods. Self-assembly and higher-order assembly in reflectin originated from a core repeating octapeptide (here named protopeptide), which may be from the same symbiotic bacteria. The origin of the reflectin gene and assembly features of reflectin protein are of considerable biological interest. The hierarchical structural architecture of reflectin and its domain and protopeptide not only provide insights for bioinspired photonic materials but also serve as unique "assembly tags" and feasible molecular platforms in biotechnology.

Reflectin as a Material for Neural Stem Cell Growth.

Cephalopods possess remarkable camouflage capabilities, which are enabled by their complex skin structure and sophisticated nervous system. Such unique characteristics have in turn inspired the design of novel functional materials and devices. Within this context, recent studies have focused on investigating the self-assembly, optical, and electrical properties of reflectin, a protein that plays a key role in cephalopod structural coloration. Herein, we report the discovery that reflectin constitutes an effective material for the growth of human neural stem/progenitor cells. Our findings may hold relevance both for understanding cephalopod embryogenesis and for developing improved protein-based bioelectronic devices.

The structure-function relationships of a natural nanoscale photonic device in cuttlefish chromatophores.

Cuttlefish, Sepia officinalis, possess neurally controlled, pigmented chromatophore organs that allow rapid changes in skin patterning and coloration in response to visual cues. This process of adaptive coloration is enabled by the 500% change in chromatophore surface area during actuation. We report two adaptations that help to explain how colour intensity is maintained in a fully expanded chromatophore when the pigment granules are distributed maximally: (i) pigment layers as thin as three granules that maintain optical effectiveness and (ii) the presence of high-refractive-index proteins-reflectin and crystallin-in granules. The latter discovery, combined with our finding that isolated chromatophore pigment granules fluoresce between 650 and 720 nm, refutes the prevailing hypothesis that cephalopod chromatophores are exclusively pigmentary organs composed solely of ommochromes. Perturbations to granular architecture alter optical properties, illustrating a role for nanostructure in the agile, optical responses of chromatophores. Our results suggest that cephalopod chromatophore pigment granules are more complex than homogeneous clusters of chromogenic pigments. They are luminescent protein nanostructures that facilitate the rapid and sophisticated changes exhibited in dermal pigmentation.

Experimental determination of refractive index of condensed reflectin in squid iridocytes.

Loliginid squid dynamically tune the structural iridescence of cells in their skin for active camouflage and communication. Bragg reflectors in these cells consist of membrane-bound lamellae periodically alternating with low refractive index extracellular spaces; neuronal signalling induces condensation of the reflectin proteins that fill the lamellae, consequently triggering the expulsion of water. This causes an increase in refractive index within the lamellae, activating reflectance, with the change in lamellar thickness and spacing progressively shifting the wavelength of reflected light. We used micro-spectrophotometry to measure the functionally relevant refractive index of the high-index lamellae of the Bragg reflectors containing the condensed reflectins in chemically fixed dermal iridocytes of the squid, Doryteuthis opalescens. Our high-magnification imaging spectrometer allowed us to obtain normalized spectra of optically distinct sections of the individual, subcellular, multi-layer Bragg stacks. Replacement of the extracellular fluid with liquids of increasing refractive index allowed us to measure the reflectivity of the Bragg stacks as it decreased progressively to 0 when the refractive index of the extracellular medium exactly matched that of the reflectin-filled lamellae, thus allowing us to directly measure the refractive index of the reflectin-filled lamellae as ncondensed lamellae ≈ 1.44. The measured value of the physiologically relevant ncondensed lamellae from these bright iridocytes falls within the range of values that we recently determined by an independent optical method and is significantly lower than values previously reported for dehydrated and air-dried reflectin films. We propose that this directly measured value for the refractive index of the squid's Bragg lamellae containing the condensed reflectins is most appropriate for calculations of reflectivity in similar reflectin-based high-index layers in other molluscs.

Optical parameters of the tunable Bragg reflectors in squid.

Cephalopods (e.g. octopus, squid and cuttlefish) dynamically tune the colour and brightness of their skin for camouflage and communication using specialized skin cells called iridocytes. We use high-resolution microspectrophotometry to investigate individual tunable Bragg structures (consisting of alternating reflectin protein-containing, high-refractive index lamellae and low-refractive index inter-lamellar spaces) in live and chemically fixed iridocytes of the California market squid, Doryteuthis opalescens. This subcellular, single-stack microspectrophotometry allows for spectral normalization, permitting use of a transfer-matrix model of Bragg reflectance to calculate all the parameters of the Bragg stack-the refractive indices, dimensions and numbers of the lamellae and inter-lamellar spaces. Results of the fitting analyses show that eight or nine pairs of low- and high-index layers typically contribute to the observed reflectivity in live cells, whereas six or seven pairs of low- and high-index layers typically contribute to the reflectivity in chemically fixed cells. The reflectin-containing, high-index lamellae of live cells have a refractive index proportional to the peak reflectivity, with an average of 1.405 ± 0.012 and a maximum around 1.44, while the reflectin-containing lamellae in fixed tissue have a refractive index of 1.413 ± 0.015 suggesting a slight increase of refractive index in the process of fixation. As expected, incremental changes in refractive index contribute to the greatest incremental changes in reflectivity for those Bragg stacks with the most layers. The excursions in dimensions required to tune the measured reflected wavelength from 675 (red) to 425 nm (blue) are a decrease from ca 150 to 80 nm for the high-index lamellae and from ca 120 to 50 nm for the low-index inter-lamellar spaces. Fixation-induced dimensional changes also are quantified, leading us to suggest that further microspectrophotometric analyses of this iridocyte system can be used as a model system to quantify the effects of various methods of tissue fixation. The microspectrophotometry technique described can be expected to provide deeper insights into the molecular and physical mechanisms governing other biophotonically active cells and structures.