Molecular mechanism of reflectin’s tunable biophotonic control: Opportunities and limitations for new optoelectronics

Abstract

Discovery that reflectin proteins fill the dynamically tunable Bragg lamellae in the reflective skin cells of certain squids has prompted efforts to design new reflectin-inspired systems for dynamic photonics. But new insights into the actual role and mechanism of action of the reflectins constrain and better define the opportunities and limitations for rationally designing optical systems with reflectin-based components. We and our colleagues have discovered that the reflectins function as a signal-controlled molecular machine, regulating an osmotic motor that tunes the thickness, spacing, and refractive index of the tunable, membrane-bound Bragg lamellae in the iridocytes of the loliginid squids. The tunable reflectin proteins, characterized by a variable number of highly conserved peptide domains interspersed with positively charged linker segments, are restricted in intra- and inter-chain contacts by Coulombic repulsion. Physiologically, this inhibition is progressively overcome by charge-neutralization resulting from acetylcholine (neurotransmitter)-induced, site-specific phosphorylation, triggering the simultaneous activation and progressive tuning of reflectance from red to blue. Details of this process have been resolved through in vitro analyses of purified recombinant reflectins, controlling charge-neutralization by pH-titration or mutation as surrogates for the in vivo phosphorylation. Results of these analyses have shown that neutralization overcoming the Coulombic inhibition reversibly and cyclably triggers condensation and secondary folding of the reflectins, with the emergence of previously cryptic, phase-segregated hydrophobic domains enabling hierarchical assembly. This tunable, reversible, and cyclable assembly regulates the Gibbs-Donnan mediated osmotic shrinking or swelling of the Bragg lamellae that tunes the brightness and color of reflected light. Our most recent studies have revealed a direct relationship between the extent of charge neutralization and the size of the reflectin assemblies, further explaining the synergistic effects on the intensity and wavelength of reflected light. Mutational analyses show that the “switch” controlling reflectins’ structural transitions is distributed along the protein, while detailed comparisons of the sequences and structures of the recently evolved tunable reflectins to those of their ancestral, non-tunable homologs are helping to identify the specific structural determinants governing tunability.

Neutralization of a Distributed Coulombic Switch Precisely Tunes Reflectin Assembly

“…These bioinformatic analyses agree with previous experimental analyses of reflectin nanoparticles by x-ray scattering, circular dichroism (CD), and Fourier-transform infrared spectroscopy (FTIR), as well as the CD analyses discussed below, all indicating that unmodified reflectins are largely disordered 6,[22][23][24][25] . Interestingly, D. opalescens reflectins A1 and A2 show overall greater predicted levels of intrinsic disorder than reflectins from other cephalopod species, which do not display tunable iridescence (Figure S1C-D) 9 . This marginally greater drive of the D. opalescens reflectins towards disorder encoded within their sequences may play a role in enabling tunable assembly by shifting the equilibrium in vivo between the monomeric and assembled states, favoring disassembly in the absence of a triggering stimulus 9 .…”

“…The tunable reflectins from the Loliginid squid Doryteuthis opalescens are essentially block-copolymeric, composed of unique and highly conserved peptide domains alternating with weakly polycationic linkers ( Figure 1A and Figure S1A) 9 . Secondary structure algorithms do not predict significant alpha or beta structure, indicating that the reflectins are intrinsically disordered 4,5 .…”

“…Although the structural reflectors of the iridocytes and leucophores of most cephalopods are static, those in the Loliginid squid family uniquely possess reversibly tunable versions of these reflectors 9 . Ultrastructural characterization of unactivated tunable iridocytes showed their intracellular lamellae to contain a heterogeneity of discontinuous ~10-20 nm nanoparticles and nanofibrils, suggesting that the cationic reflectins within exist in a predominantly unassembled state dominated by inter-particle charge repulsion 9,10 .…”

“…Although the structural reflectors of the iridocytes and leucophores of most cephalopods are static, those in the Loliginid squid family uniquely possess reversibly tunable versions of these reflectors 9 . Ultrastructural characterization of unactivated tunable iridocytes showed their intracellular lamellae to contain a heterogeneity of discontinuous ~10-20 nm nanoparticles and nanofibrils, suggesting that the cationic reflectins within exist in a predominantly unassembled state dominated by inter-particle charge repulsion 9,10 . However, upon iridocyte activation initiated by binding of the neurotransmitter acetylcholine (ACh), released from nearby nerve cells, to cell surface muscarinic receptors, a signal transduction cascade culminates in enzymatic phosphorylation of the reflectins, consequently neutralizing their cationic charge and driving assembly of the reflectins to form homogenously densely staining Bragg lamellae 5,7,10 .…”

“…The reflectins are essentially block copolymers, being composed of highly conserved reflectin domains (RMs, for "Reflectin Motifs") interspersed with cationic linkers, as seen for the reflectins A1 and A2 of the Loliginid squid Doryteuthis opalescens (Figure 1A). The reflectins exhibit a unique amino acid composition with some heterogeneity across their sequences, being highly enriched in methionine, arginine and tyrosine residues, and possess almost no (<1%) aliphatic residues 4,8,9 . The sequence composition of the reflectins suggests that attractive interactions are likely to be primarily driven by tyrosine-aromatic (pi-pi), arginine-tyrosine (cation-pi) and methionine-tyrosine (sulfurpi) interactions, forming a complex interaction network of both intra-and inter-strand non-covalent bonding 9,13 .…”