To help meet the needs for a new generation of advanced, high-performance composites required for new construction materials, new micro-electronic, optoelectronic and catalytic devices, chemical and biological sensors (Bright, 1994), energy transducers, "smart" medical implants (Newnham, 1997) and faster and higher capacity “biochips” (Kozal et al., 1996; Travis, 1997), materials scientists have turned to the biomimetic production of mineralized nanocomposites modeled on those produced by marine organisms (Eisner, 1991; Mann et al., 1992, 1993; Heuer et al., 1992; Pennisi, 1992; Mann, 1993; Walsh et al, 1994; Hoch et al., 1996; Mann and Ozin, 1996; Yang et al., 1997). These ceramic-like composites of mineral and biopolymers offer unique combinations of strength, anisotropy, biocompatibility, precise structural control at the nanoscale level, and coupling between mineral and organic polymeric phases that make them especially attractive models for commercially valuable new materials (e.g., Bouchon et al., 1995; Hoch et al., 1996).

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.

Marine sponges produce nanoscale silicon dioxide structures called spicules. In previous California Sea Grant research, UC Santa Barbara professor Daniel Morse and colleagues identified the principal protein responsible for directing spicule construction at the molecular and atomic scale.

Reflectins are a unique class of proteins found in the specialized iridescent cells, iridocytes, in cephalopods. The colors reflected by dorsal iridocytes from the California market squid Doryteuthis opalescens are neuronally tunable, and these cells are enriched in reflectins A1, A2, B, and C. The phosphorylation of cationic reflectin proteins result in reflectin assembly: the degree of assembly controls the extent of dehydration of the light-scattering Bragg lamellae, adjusting their dimensions and therefore wavelengths of light that are reflected. I show that reflectin A1 assemblies are in dynamic exchange through a dilute monomeric phase and characterize the aging of dynamic exchange using DLS (dynamic light scattering) and FRET (Forster resonant energy transfer). Reflectin A1 phase behavior is determined by protein net charge density and ionic strength. At high net charge densities, increasing salt concentration drives liquid-liquid phase separation of reflectin A1 via nanoscale assembly intermediates. The hydrophobic effect and electrostatic screening of intra- and inter-protein Coulombic repulsion progressively drive reflectin A1 folding, assembly, and LLPS. Using fluorescence recovery after photobleaching (FRAP) and droplet fusion dynamics I demonstrate the liquidity of reflectin A1 condensates is tuned by protein NCD and ionic strength. Reflectin C co-phase separates with A1 and drastically increases the liquidity of reflectin A1. Condensates formed from physiologically relevant mixtures of reflectins A1, A2, B, and C show complex layered droplet formation in which the relative miscibility of each reflectin varies with pH, ionic strength, and proportions of reflectin species. The many inputs that define multi-reflectin phase behavior in vivo shows reflectins usefulness for creating soft biomaterials with tunable spatial organizations and liquid properties. These findings also demonstrate that LLPS of reflectins A1, A2, B and C could occur upon iridocyte activation, and that ratios of these reflectin species found in tunable iridocytes could enhance the tunability of lamellar dehydration induced by reflectin phosphorylation.

Although pigments contribute much of the brilliant purple and orange coloration of the aeolid nudibranch, Flabellina iodinea, the brightness of both colors was found to be enhanced by unusually sparkling iridocytes that exhibit rapid temporal variation in the brightness of their reflectivity. Electron micrographic examination revealed these epidermal cells to contain numerous multilayer stacks of crystals, both within vesicles and apparently free in the cells. High-resolution light microscopy showed that these structures tumble freely to produce the observed sparkling reflectivity. Most abundant near the epithelial basal lamina, the perceived color of these sparking iridocytes appears dependent on the size and thickness of the crystal platelets that comprise the stacks, with those that exhibit silver reflectance in the orange cerata being larger and thicker than those that reflect blue in the epithelium of the purple body. Thin layer chromatography and UV spectrometry show that the crystals isolated from all epithelial regions are identical in composition, with guanine as the major component and its derivative, hypoxanthine, a minor component. Electron diffraction of the crystals isolated from the orange and purple tissue exhibit nearly identical lattice parameters of a = 6.57 ± 0.05 Å, b = 13.65 ± 0.07 Å and c = 18.76 ± 0.06 Å - unit cell dimensions that closely match those measured for the non-tumbling guanine crystals widely distributed in other biophotonic systems ranging from marine invertebrates to terrestrial vertebrates. The epidermal iridocytes of F. iodinea are thus capable of manipulating crystal growth to produce multilayer stacks of purine crystals with optical properties specific to different locations in the body. This specificity parallels and apparently augments the animal’s striking pattern of pigmentation.