Reversible changes in the phosphorylation of reflectin proteins have been shown to drive the tunability of color and brightness of light reflected from specialized cells in the skin of squids and related cephalopods. We show here, using dynamic light scattering, electron microscopy, and fluorescence analyses, that reversible titration of the excess positive charges of the reflectins, comparable with that produced by phosphorylation, is sufficient to drive the reversible condensation and hierarchical assembly of these proteins. The results suggest a two-stage process in which charge neutralization first triggers condensation, resulting in the emergence of previously cryptic structures that subsequently mediate reversible, hierarchical assembly. The extent to which cyclability is seen in the in vitro formation and disassembly of complexes estimated to contain several thousand reflectin molecules suggests that intrinsic sequence-and structure-determined specificity governs the reversible condensation and assembly of the reflectins and that these processes are therefore sufficient to produce the reversible changes in refractive index, thickness, and spacing of the reflectin-containing subcellular Bragg lamellae to change the brightness and color of reflected light. This molecular mechanism points to the metastability of reflectins as the centrally important design principle governing biophotonic tunability in this system. Cephalopods (squids, octopi, and cuttlefish) are well known for their diversity of light-manipulating, pigment-based, and nano-structural systems used for camouflage and underwater communication (1, 2). Of these systems, the dynamically tunable structural color of certain squids holds great interest as models for next-generation tunable optical materials and devices (3, 4). Reflectins are a class of proteins originally identified in the reflective tissue of the Hawaiian bobtail squid, Euprymna scolopes (5), and have since been found in multiple squid species, including the pelagic Pacific and Atlantic squids Doryteuthis opalescens and Doryteuthis pealeii, respectively (6 -8). In these latter two species, the reflectins constitute the principal constituents of the dynamically controlled subcellular Bragg reflector lamellae responsible for the tunable color and intensity of reflected light in "iridocyte" cells (8) and the subcellular vesicles responsible for switchable bright white Mie scattering in specialized "leucophore" cells in females of the Pacific species (the only example, to our knowledge, of switchable broadband reflectance in molluscs) (6). Reflectins are also found, although in a different molar ratio, in the static (nontunable) Bragg lamellae of fixed-color iridocytes in this species (7).Reflectance from both the tunable iridocytes and switchable leucophores is activated by the diffusion of acetylcholine (ACh) 2 (8, 9), recently discovered to be released from fine neuronal processes innervating local areas of the squid skin (10). In the tunable iridocytes, it has been shown that the act...

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.

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

Summary The interface between the membrane (MS) and cytoplasmic (C) rings of the bacterial flagellar motor couples torque generation to rotation within the membrane. The structure of the C-terminal helices of the integral membrane protein FliF (FliFC) bound to the N-terminus of the switch complex protein FliG (FliGN) reveals that FliGN folds around FliFC to produce a topology that closely resembles both the middle and C-terminal domains of FliG. The interface is consistent with solution-state NMR, SAXS, in vivo interaction studies and cellular motility assays. Co-folding with FliFC induces substantial conformational changes in FliGN, and suggests that FliF and FliG have the same stoichiometry within the rotor. Modeling the FliFC:FliGN complex into cryoEM rotor density updates the architecture of the middle and upper switch complex and shows how domain shuffling of a conserved interaction module anchors the cytoplasmic rotor to the membrane.

The binding of the soluble cytoplasmic protein FliG to the transmembrane protein FliF is one of the first interactions in the assembly of the bacterial flagellum. Once established, this interaction is integral in keeping the flagellar cytoplasmic ring, responsible for both transmission of torque and control of the rotational direction of the flagellum, anchored to the central transmembrane ring on which the flagellum is assembled. Here we isolate and characterize the interaction between the N-terminal domain of Thermotoga maritima FliG (FliGN) and peptides corresponding to the conserved C-terminal portion of T. maritima FliF. Using nuclear magnetic resonance and other techniques we show that the last ~40 amino acids of FliF (FliF) interac strongly (upper-bound Kd of low nM) with FliGN. This complex formation causes extensive conformational changes in FliGN. We find that T. maritima FliGN is homodimeric in the absence of FliFC peptide but forms a heterodimeric complex with peptide, and we show that this same change in oligomeric state occurs in full-length T. maritima FliG as well. We relate previously observed phenotypic effects of FliFC mutations to our direct observation of binding. Lastly, based on NMR data we propose that the primary interaction site for FliFC is located on a conserved hydrophobic patch centered along helix 1 of FliGN. These results provide new detailed information about the bacterial flagellar motor and support efforts to understand the cytoplasmic ring’s precise molecular structure and mechanism of rotational switching.