Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color... more Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing its refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that reduction in repulsive net charge of the purified, recombinant reflectin – either (for the first time) by generalized anionic screening with salt, or by pH titration - drives a finely tuned, precisely calibrated increase in size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. X-ray scattering analyses confirm the spheric...
Cephalopods possess a sophisticated array of mechanisms to achieve camouflage in dynamic underwat... more Cephalopods possess a sophisticated array of mechanisms to achieve camouflage in dynamic underwater environments. While active mechanisms such as chromatophore patterning and body posturing are well known, passive mechanisms such as manipulating light with highly evolved reflectors may also play an important role. To explore the contribution of passive mechanisms to cephalopod camouflage, we investigated the optical and biochemical properties of the silver layer covering the eye of the California fishery squid,Loligo opalescens. We discovered a novel nested-spindle geometry whose correlated structure effectively emulates a randomly distributed Bragg reflector (DBR), with a range of spatial frequencies resulting in broadband visible reflectance, making it a nearly ideal passive camouflage material for the depth at which these animals live. We used the transfer-matrix method of optical modelling to investigate specular reflection from the spindle structures, demonstrating that a DBR w...
Reflectin proteins are widely distributed in reflective structures in cephalopods, but only in Lo... more 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...
In this work, we demonstrate electrochemical triggering and assembly of reflectin, the protein th... more In this work, we demonstrate electrochemical triggering and assembly of reflectin, the protein that mediates neuronal fine-tuning of color reflected from skin cells used for camouflage and communication in squids. Electrochemical reduction of histidine residues in the protein acts as an in vitro surrogate for phosphorylation in vivo, driving the assembly previously shown to regulate its function. Using micro-drop voltammetry and in situ dynamic light scattering, we demonstrate selective reduction of the imidazolium sidechains of histidine in monomers, oligopeptides and reflectin in acidic 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 reflectin. The resulting “electro-assembly” methodology provides a new approach to probe, understand, and control the mechanisms that dynamically tune protein struc...
Journal of the Royal Society, Interface / the Royal Society, Jul 1, 2016
A surprising recent discovery revealed that the brightly reflective cells ('iridocytes') ... more A surprising recent discovery revealed that the brightly reflective cells ('iridocytes') in the epithelia of giant clams actually send the majority of incident photons 'forward' into the tissue. While the intracellular Bragg reflectors in these cells are responsible for their colourful back reflection, Mie scattering produces the forward scattering, thus illuminating a dense population of endosymbiotic, photosynthetic microalgae. We now present a detailed micro-spectrophotometric characterization of the Bragg stacks in the iridocytes in live tissue to obtain the refractive index of the high-index layers (1.39 to 1.58, average 1.44 ± 0.04), the thicknesses of the high- and low-index layers (50-150 nm), and the numbers of pairs of layers (2-11) that participate in the observed spectral reflection. Based on these measurements, we performed electromagnetic simulations to better understand the optical behaviour of the iridocytes. The results open a deeper understanding of...
Reversible changes in phosphorylation of the reflectin proteins have been shown to drive the tuna... more Reversible changes in phosphorylation of the 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 to that produced by phosphorylation, is sufficient to drive the reversible condensation and hierarchical assembly of these proteins. 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 co...
SUMMARY There are dramatic and physiologically relevant changes in both skylight color and intens... more SUMMARY There are dramatic and physiologically relevant changes in both skylight color and intensity during evening twilight as the pathlength of direct sunlight through the atmosphere increases, ozone increasingly absorbs long wavelengths and skylight becomes increasingly blue shifted. The moon is above the horizon at sunset during the waxing phase of the lunar cycle, on the horizon at sunset on the night of the full moon and below the horizon during the waning phase. Moonlight is red shifted compared with daylight, so the presence, phase and position of the moon in the sky could modulate the blue shifts during twilight. Therefore, the influence of the moon on twilight color is likely to differ somewhat each night of the lunar cycle, and to vary especially rapidly around the full moon, as the moon transitions from above to below the horizon during twilight. Many important light-mediated biological processes occur during twilight, and this lunar effect may play a role. One particula...
ABSTRACT A semiconducting lanthanum-doped barium titanate ceramic has been fabricated for battery... more ABSTRACT A semiconducting lanthanum-doped barium titanate ceramic has been fabricated for battery safety applications by simple means from nanoparticles prepared at room temperature by kinetically controlled vapor diffusion catalysis. The material, characterized by electron microscopy, X-ray diffraction and electrical measurements, exhibits a difficult to achieve combination of submicron grain size (∼500nm) and attractive electrical properties of room temperature resistivity below 100Ωcm and a 12-fold increase in resistivity through the Curie temperature (positive thermal coefficient of resistivity, PTCR). Systematic investigation of sintering conditions revealed that a short period of heating at 1350°C under air is necessary to suppress abnormal grain growth, while precise control of the cooling rate is needed to achieve the targeted electrical properties. Cooling must be sufficiently fast to avoid complete back-oxidation, yet slow enough to facilitate oxygen adsorption at the grain boundaries to produce the thin oxide layer apparently responsible for the observed PTCR.
ABSTRACT (Figure Presented) Enzymatic polymer synthesis is of considerable interest because of th... more ABSTRACT (Figure Presented) Enzymatic polymer synthesis is of considerable interest because of the synthetic advantages afforded by biological catalysts. Silicatein enzymes have shown broad synthetic utility for inorganic materials, and recombinant and native silicateins have been applied in the preparation of poly(L-lactide) (PLA). The picture shows native silicatein filaments coated with PLA (scale bar = 4 μm).
We recently discovered the molecular mechanism by which a common California marine sponge synthes... more We recently discovered the molecular mechanism by which a common California marine sponge synthesizes the fiberglass-like structures that form the internal skeleton of this simple marine animal. Our goals are: (1) to harness the proteins and molecular mechanisms that control this synthesis to make valuable semiconductors inexpensively under environmentally friendly conditions; and (2) to use this new approach to develop, low-cost, high-efficiency solar energy converters and other practical electronic applications. Training of students in the unique interdisciplinary approach of this project, combining new developments in biotechnology, advanced instrumentation, and materials engineering to produce advanced high-performance materials, also is a major objective of our project.
Nature is the international weekly journal of science: a magazine style journal that publishes fu... more Nature is the international weekly journal of science: a magazine style journal that publishes full-length research papers in all disciplines of science, as well as News and Views, reviews, news, features, commentaries, web focuses and more, covering all branches of science and ...
Phosphorylation is among the most widely distributed mechanisms regulating the tunable structure ... more 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 e...
Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color... more Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing its refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that reduction in repulsive net charge of the purified, recombinant reflectin – either (for the first time) by generalized anionic screening with salt, or by pH titration - drives a finely tuned, precisely calibrated increase in size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. X-ray scattering analyses confirm the spheric...
Cephalopods possess a sophisticated array of mechanisms to achieve camouflage in dynamic underwat... more Cephalopods possess a sophisticated array of mechanisms to achieve camouflage in dynamic underwater environments. While active mechanisms such as chromatophore patterning and body posturing are well known, passive mechanisms such as manipulating light with highly evolved reflectors may also play an important role. To explore the contribution of passive mechanisms to cephalopod camouflage, we investigated the optical and biochemical properties of the silver layer covering the eye of the California fishery squid,Loligo opalescens. We discovered a novel nested-spindle geometry whose correlated structure effectively emulates a randomly distributed Bragg reflector (DBR), with a range of spatial frequencies resulting in broadband visible reflectance, making it a nearly ideal passive camouflage material for the depth at which these animals live. We used the transfer-matrix method of optical modelling to investigate specular reflection from the spindle structures, demonstrating that a DBR w...
Reflectin proteins are widely distributed in reflective structures in cephalopods, but only in Lo... more 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...
In this work, we demonstrate electrochemical triggering and assembly of reflectin, the protein th... more In this work, we demonstrate electrochemical triggering and assembly of reflectin, the protein that mediates neuronal fine-tuning of color reflected from skin cells used for camouflage and communication in squids. Electrochemical reduction of histidine residues in the protein acts as an in vitro surrogate for phosphorylation in vivo, driving the assembly previously shown to regulate its function. Using micro-drop voltammetry and in situ dynamic light scattering, we demonstrate selective reduction of the imidazolium sidechains of histidine in monomers, oligopeptides and reflectin in acidic 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 reflectin. The resulting “electro-assembly” methodology provides a new approach to probe, understand, and control the mechanisms that dynamically tune protein struc...
Journal of the Royal Society, Interface / the Royal Society, Jul 1, 2016
A surprising recent discovery revealed that the brightly reflective cells ('iridocytes') ... more A surprising recent discovery revealed that the brightly reflective cells ('iridocytes') in the epithelia of giant clams actually send the majority of incident photons 'forward' into the tissue. While the intracellular Bragg reflectors in these cells are responsible for their colourful back reflection, Mie scattering produces the forward scattering, thus illuminating a dense population of endosymbiotic, photosynthetic microalgae. We now present a detailed micro-spectrophotometric characterization of the Bragg stacks in the iridocytes in live tissue to obtain the refractive index of the high-index layers (1.39 to 1.58, average 1.44 ± 0.04), the thicknesses of the high- and low-index layers (50-150 nm), and the numbers of pairs of layers (2-11) that participate in the observed spectral reflection. Based on these measurements, we performed electromagnetic simulations to better understand the optical behaviour of the iridocytes. The results open a deeper understanding of...
Reversible changes in phosphorylation of the reflectin proteins have been shown to drive the tuna... more Reversible changes in phosphorylation of the 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 to that produced by phosphorylation, is sufficient to drive the reversible condensation and hierarchical assembly of these proteins. 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 co...
SUMMARY There are dramatic and physiologically relevant changes in both skylight color and intens... more SUMMARY There are dramatic and physiologically relevant changes in both skylight color and intensity during evening twilight as the pathlength of direct sunlight through the atmosphere increases, ozone increasingly absorbs long wavelengths and skylight becomes increasingly blue shifted. The moon is above the horizon at sunset during the waxing phase of the lunar cycle, on the horizon at sunset on the night of the full moon and below the horizon during the waning phase. Moonlight is red shifted compared with daylight, so the presence, phase and position of the moon in the sky could modulate the blue shifts during twilight. Therefore, the influence of the moon on twilight color is likely to differ somewhat each night of the lunar cycle, and to vary especially rapidly around the full moon, as the moon transitions from above to below the horizon during twilight. Many important light-mediated biological processes occur during twilight, and this lunar effect may play a role. One particula...
ABSTRACT A semiconducting lanthanum-doped barium titanate ceramic has been fabricated for battery... more ABSTRACT A semiconducting lanthanum-doped barium titanate ceramic has been fabricated for battery safety applications by simple means from nanoparticles prepared at room temperature by kinetically controlled vapor diffusion catalysis. The material, characterized by electron microscopy, X-ray diffraction and electrical measurements, exhibits a difficult to achieve combination of submicron grain size (∼500nm) and attractive electrical properties of room temperature resistivity below 100Ωcm and a 12-fold increase in resistivity through the Curie temperature (positive thermal coefficient of resistivity, PTCR). Systematic investigation of sintering conditions revealed that a short period of heating at 1350°C under air is necessary to suppress abnormal grain growth, while precise control of the cooling rate is needed to achieve the targeted electrical properties. Cooling must be sufficiently fast to avoid complete back-oxidation, yet slow enough to facilitate oxygen adsorption at the grain boundaries to produce the thin oxide layer apparently responsible for the observed PTCR.
ABSTRACT (Figure Presented) Enzymatic polymer synthesis is of considerable interest because of th... more ABSTRACT (Figure Presented) Enzymatic polymer synthesis is of considerable interest because of the synthetic advantages afforded by biological catalysts. Silicatein enzymes have shown broad synthetic utility for inorganic materials, and recombinant and native silicateins have been applied in the preparation of poly(L-lactide) (PLA). The picture shows native silicatein filaments coated with PLA (scale bar = 4 μm).
We recently discovered the molecular mechanism by which a common California marine sponge synthes... more We recently discovered the molecular mechanism by which a common California marine sponge synthesizes the fiberglass-like structures that form the internal skeleton of this simple marine animal. Our goals are: (1) to harness the proteins and molecular mechanisms that control this synthesis to make valuable semiconductors inexpensively under environmentally friendly conditions; and (2) to use this new approach to develop, low-cost, high-efficiency solar energy converters and other practical electronic applications. Training of students in the unique interdisciplinary approach of this project, combining new developments in biotechnology, advanced instrumentation, and materials engineering to produce advanced high-performance materials, also is a major objective of our project.
Nature is the international weekly journal of science: a magazine style journal that publishes fu... more Nature is the international weekly journal of science: a magazine style journal that publishes full-length research papers in all disciplines of science, as well as News and Views, reviews, news, features, commentaries, web focuses and more, covering all branches of science and ...
Phosphorylation is among the most widely distributed mechanisms regulating the tunable structure ... more 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 e...
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