Elucidating the Role of Nanostructured Pigment Granules in the Dynamic Coloration of Cephalopods: A Pathway Towards New Bioinspired Materials

by Thomas L. Williams

B.A. in Chemistry, Colby College

A dissertation submitted to

The Faculty of the College of Science of Northeastern University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

February 14th, 2019

Dissertation directed by

Leila Deravi Professor of Chemistry and Chemical Biology

1 Acknowledgements

First, I would like to sincerely extend my gratitude to my advisor, Prof. Leila Deravi, for an incredible amount of direction, advice, and patience.

I would also like to thank my dissertation committee members: Profs. Steven Lopez, Eugene Smotkin, and Rudi Seitz. Each of you have contributed significantly to this work and to my growth as a chemist through your comments and guidance.

Thanks to my family, who have always been supportive of my academic endeavors.

I also need to extend my thanks to all the current and previous members of the Biomaterials Design Group, especially Christopher Dibona, Dr. Sean Dinneen, Dr. Amrita Kumar, and Camille Martin, aka Team Pigment, for their assistance, advice and support. I would also like to thank Conor Gomes, Cassandra Martin, and Dr. Jeff Paten for their insight and friendship.

I would also like to thank Northeastern University and the Department of Chemistry and Chemical Biology for the opportunity to work and study here. I would like to specifically thank Prof. Jared Auclair and Dr. John de la Parra for their assistance with mass spectrometry, as well as Prof. Jason Guo, for his assistance with NMR. I also am grateful to Emanuelle Hestermann, Paige Scannell, Rich Pumphrey and Tara Loschiavo.

Thanks to the faculty and staff at the Marine Biological Laboratory, especially Dr. Roger Hanlon and Dr. Stephen Senft, for their collaboration and hospitality. Without their work, my own would lack biological context.

I would also like to thank my friends, coworkers, and mentors at the University of New Hampshire.

Finally, thanks for funding goes to the National Science Foundation (DMR- 1700720), the Barnett Institute of Chemical and Biological Analysis, and the Department of Chemistry and Chemical Biology at Northeastern University.

2 Abstract of Dissertation

Cephalopods such as squid, octopus, and cuttlefish can quickly and accurately modulate their coloration in order to camouflage themselves to avoid predators, hunt prey, or communicate within their natural environment. This ability is made possible, in part, by chromatophores: pigmentary organs that can expand or relax to impart a global color change to the animal. While it is known that visible color originates from the nanostructured pigment granules that populate the chromatophore, their compositional and structural contributions to coloration remain largely unknown. The overarching goal of this dissertation is to elucidate the structure and function of the cephalopod chromatophore pigment granules as a means to inspire the design of new advanced optical materials.

First, the presence of ommochrome pigments within the chromatophore granules was confirmed, and these pigments were identified as xanthommatin and decarboxylated xanthommatin. In this process, the pigments were extracted from the granules of isolated squid

Doryteuthis pealeii chromatophores, resulting in a 70.8% decrease in the diameter of the granules, as well as a loss of absorbance, suggesting that non-pigmentary structural components, likely proteins, contribute to granule structure as well. In the same study, we examined the effect of granule structure on the optical properties of the pigments, finding that the extracted pigment features a blue shift in maximum absorbance as well as an altered fluorescence profile compared to the intact granules, suggesting that the optical properties of chromatophore pigment granules arise from an interaction between the pigmentary and structural components.

Next, we performed a proteomic study of chromatophores and pigment granules isolated from D. pealeii dermal tissue to determine what proteins are in the granules that may also

3 contribute to coloration. Lens crystallin proteins, specifically Ω- and S-crystallin isoforms, were found in the pigment granules, suggesting that the interaction between the pigment and these high- refractive index proteins may enhance absorbance through scattering effects. Reflectin, a cephalopod-specific protein responsible for structural coloration, was also identified in chromatophores, but not within the pigment granules. This finding inspired a re-examination of the chromatophore, leading to the conclusion that these organs incorporate elements of protein- based structural coloration, likely through thin-film interference that may amplify the colors displayed by the animal, originating from reflectin in sheath cells in the periphery of the chromatophore.

Finally, a new, scalable method for the synthesis of xanthommatin, the primary pigment confined to the chromatophore granules, was developed and characterized. By replacing the traditionally used oxidizing agent with an applied electrochemical potential, the reaction becomes simpler while retaining its kinetics, specificity, and yield. Computational chemistry, informed by previous literature, was used to determine the mechanisms underlying the reaction so that it can more easily be applied to the synthesis of xanthommatin derivatives. Together, these results indicate a possible pathway through which the production of asymmetric phenoxazinones might be scaled up for future materials applications.

4 Table of Contents Acknowledgements 2 Abstract of Dissertation 3 Table of Contents 5 List of Figures 7 List of Tables 8 Chapter 1: Introduction to Cephalopod-Inspired Materials 9 1.1 Bioinspired Design 9 1.2 Cephalopods and Dynamic Coloration 11 1.3 Iridophores 13 1.4 Leucophores 19 1.5 Chromatophores 21 1.6 Ommochromes and Xanthommatin 26 1.7 Pigment Granules in Insects 31 1.8 Reflectin and Crystallin 33 1.9 Cephalopod Inspired Materials 37 1.10 Dissertation Aims 42 Chapter 2: Overview of Methods 44 2.1 Animal Collection 44 2.2 Chromatophore Isolation 44 2.3 Collection of Chromatophore Granules 44 2.4 Pigment Extraction 45 2.5 Pigment Separation 45 2.6 Scanning Electron Microscopy 46 2.7 Ultraviolet-Visible Light Spectroscopy 46 2.8 Fluorescence Spectroscopy 47 2.9 Mass Spectrometry 47 2.10 Isolation of Chromatophores for Whole Cell Analysis 48 2.11 Protein Purification and Analysis Using Mass Spectrometry 48 2.12 Sample Collection and RNA Sequencing of Squid Chromatophore 50 2.13 Transcriptome Assembly 51 2.14 Protein Identification using BLAST 51 2.15 Spectral Count Data Processing 52 2.16 Synthesis of Xanthommatin 53 2.17 Electrosynthesis of Xanthommatin 53 2.18 Characterization of Electrosynthesized Xanthommatin 54 2.19 Confirmation of Electrosynthesized Xanthommatin Structure 54 2.20 Computational Methods 55

5 Chapter 3: Contributions of Phenoxazone-Based Pigments to the Structure 53 and Function of Nanostructured Granules in Squid Chromatophores 3.1 Introduction 53 3.2 Results and Discussion 58 3.3 Conclusions 68

Chapter 4: Dynamic Pigmentary and Structural Coloration Within 70 Cephalopod Chromatophore Organs 4.1 Introduction 70 4.2 Results 4.2.1. Expanded Chromatophores Exhibit Intense Structural Color 72 4.2.2 Composition Analysis of the Squid Chromatophore 74 4.2.3. Crystallin But Not Reflectin Is Found Amidst Pigment Granules 78 4.2.4. Reflectin Molecules Are Distributed Throughout Sheath Cells 80 4.2 Discussion 83 Chapter 5: A scalable method to synthesize the xanthommatin biochrome 89 via an electro catalyzed oxidation of tryptophan metabolites 5.1 Introduction 89 5.2 Results 91 5.3 Conclusions 101

Chapter 6: Conclusions and Recommendations 102 References 105 Appendices 117 A1. Proteomics Data from Chromatophores and Pigment Granules 117 A2. Computational Data 159 A2.1. Coordinates and energies of the closed-shell substitution reaction 159 A2.2. Coordinates and energies of the open-shell substitution reaction 163 A2.3. Coordinates and energies of the closed-shell addition reaction 166 A2.4. Coordinates and energies of the open-shell addition reaction 171 A2.5. Coordinates and energies of the final product, H2Xa 175 A2.6. Coordinates and energies of the leaving groups 178

6 List of Figures

Figure 1.1. Photographs of cephalopods exhibiting dynamic coloration 11 Figure 1.2. Male cuttlefish performing the Intense Zebra Display 12 Figure 1.3. Cross-sectional TEM Image of Octopus dofleini dermis 14 Figure 1.4. Multilayer interference diagram and simulated effect 16 Figure 1.5. Illustration of tunable iridophore mechanism 18 Figure 1.6. Reflectance spectra and images of switchable iridophores 20 Figure 1.7. Drawing of cephalopod chromatophore organ 22 Figure 1.8. Examples of ommochromes derived from xanthommatin 26 Figure 1.9. Biochemical synthesis of xanthommatin 29 Figure 1.10. Kynurenine monooxygenase distribution in nature 31 Figure 1.11. Proposed model of ommochromosome formation in spiders 33 Figure 1.12. Repeated peptides in and overall composition of reflectin 36 Figure 1.13. Examples of cephalopod inspired technology 40

Figure 2.1 SDS-PAGE of proteins collected from whole chromatophores 49 Figure 2.2 SDS-PAGE of proteins from isolated granules, pigment, and 50 extracted granules

Figure 3.1. Results of pigment extraction from granules 59 Figure 3.2. Separation of pigments by preparative TLC 61 Figure 3.3. 3D Fluorescence spectra of granules and pigments 63 Figure 3.4. Mass spectral analysis of extracted pigment composition 65 Figure 3.5. Electrochemical characterization of xanthommatin 67

Figure 4.1. Iridescence produced in chromatophores of live squid 73 Figure 4.2. Mass spectrometric analysis of chromatophore proteins 76 Figure 4.3. QSpec significance analysis of chromatophore proteins 78 Figure 4.4. Localization of reflectin to sheath cells 81 Figure 4.5. Illustration of light interactions in squid dermis 87

Figure 5.1. Summary of xanthommatin electrosynthesis 92 Figure 5.2. Spectrometric and voltammetric characterization electrosynthesis 93 Figure 5.3. NMR spectra of electrosynthetic xanthommatin 94 Figure 5.4. Controlled-potential chronocoulometry of the scaled-up reaction 95 Figure 5.5 A proposed mechanism of 3-OHK dimerization 97 Figure 5.6. Calculated energy barriers xanthommatin cyclization 99 Figure 5.7. Calculated geometry of transition states in xanthommatin cyclization 100

7 List of Tables

Table 1.1. Optical properties of extracted pigments under various conditions 23

Table A.1. Normalized Peptide Counts and Identities of Detected Proteins in 116 Whole Chromatophores

Table A.2. Normalized Peptide Counts and Identities of Detected Proteins in 152 Isolated Pigment Granules

Table A.3. Normalized Peptide Counts and Identities of Detected Proteins in 156 Pigment-Extracted Granules

Table A.4. Normalized Peptide Counts and Identities of Detected Proteins in 157 Pigment

8 Chapter 1: Introduction to Cephalopod-inspired Materials 1.1 Bioinspired Design As we require more sophisticated solutions to modern problems, we look to natural systems for inspiration. Bio-inspired design is the process of studying biological phenomena and applying the underlying principles to the development of new technology. Because natural selection drives the optimization of biological systems, studying these systems can provide insight into simple and efficient design to achieve complex function.1 In terms of chemistry, bioinspired design tends to overlap with the principles green chemistry, as biological systems use aqueous, rather than organic, solvents and tend to produce incredibly complexity from simple, sustainable starting materials using specific and efficient catalysts. Solar fuel production is one example of this: the production of useful carbon compounds from carbon dioxide (CO2) using solar power is directly parallel to photosynthesis in plants. In plants, antenna complexes, comprising chlorophyll pigments and proteins, absorb light and transfer the energy to the reaction center, where a central chlorophyll reduces pheophytin, the first step on the electron transport chain, and oxidizes water in order to regenerate the active state.2 Efficiently replicating this process would provide an avenue for the production of a carbon neutral fuel, requiring only water and CO2, a pollutant, as reagents and light as a driving force.2-3

Another area in which biological systems inspire materials development is the production of nanoscale silica structures based on diatoms. Diatoms have a silica-based cell wall, called a frustule, produced by the condensation of silica on proteins.4 This condensation relies on the interaction between silicic acid and long chain polyamines, such as are found in the post- translationally modified silaffin proteins.5-6 This reaction has been replicated with amine-rich dendrimers that provide a synthetic substrate for silica condensation, as well as with non-native proteins, such as human fibronectin.7-8 Biogenic silica produced by diatoms already has

9 applications in drug delivery, chemical separations, and novel sensors, but by replicating the silica deposition process with customizable substrates, new materials with more desirable attributes can be developed.4, 6

For this work, we will consider the ability of cephalopods to dynamically alter their coloration in order to produce impressive visual displays as well as intricate disguises (Figure

1.1).9-11 This ability was noted by Aristotle in his History of Animals in the 4th century BCE, and has continued to fascinate the general public as well as scientists up to the present day.12 The ability to replicate this dynamic coloration would have obvious implications for the development of adaptive camouflage, but, in a more general sense, bioinspired technology based on this dynamic coloration has applications in flexible displays and nanophotonics.13-14 However, before new technologies can be developed from these systems, there must be a better understanding of the mechanisms underlying this ability.

10

Figure 1.1. An example of coloration in a squid and a variety of different cuttlefish colorations. (a) Disruptive bars and iridescent spots in D. Pealeii. (b) A cuttlefish, Sepia apama, exhibiting blue-green iridescence with white stripes. (c) S. apama in a camouflaged state (d) S. apama showing brown coloration.15 Reproduced from Ref. 15.

1.2 Cephalopods and Dynamic Coloration

Cephalopods, are a class of mollusk including octopus, cuttlefish, and squid.16 Species from all three of these broad categories express some level of dynamic coloration, and while there are differences in the extent of this ability, the fact that it is conserved across orders suggests that many of the basic mechanisms should also be well conserved, at least on the cellular level.15-16

Before looking at these cellular mechanisms, though, it is worth examining how this dynamic coloration is used by these organisms to avoid detection or to communicate.

11 When threatened, Sepia officinalis, the European cuttlefish, has been shown to use this dynamic coloration to produce eye-spots, known as a deimatic display, to startle certain predators that depend on visual cues.17 Another example of this is Thaumoctopus mimicus, the mimic octopus, which uses this dynamic coloration to produce striking white and black bands, allowing it to take on the appearance of other, more dangerous animals, such as banded sea-snakes and lion- fishes, to deter predators.18 Cephalopods have been observed altering their appearance to both blend in with their background via dynamic camouflage, as shown in Figures 1.1a and 1.1c, as well as to disguise themselves as inanimate objects, like rocks, a technique known as masquerade.10

Figure 1.2. Two male cuttlefish (S. officinalis) performing the Intense Zebra Display. The animal on the left is showing a light “face,” indicating that it does not intend to attack. The arrows indicate the “extended fourth arm,” which accompanies the zebra body pattern in the Intense Zebra Display.19 Reproduced from Ref. 19. This dynamic coloration also plays a role in communication between individual cephalopods. A study featuring S. officinalis examined a specific coloration pattern, termed the

“Intense Zebra Display,” which is specific to the male cuttlefish, (Figure 1.2). This pattern not

12 only allows male cuttlefish to identify each other but also these male cuttlefish can modulate the darkness of their face to indicate whether they wish to attack.19 These displays, as well as acts of camouflage, masquerade, and deimatic display, require cuttlefish to examine situations and weigh decisions in order to apply the correct visual and textural modulations.10, 17, 19

In contrast to chameleons, which also famously feature the ability to alter coloration, cephalopods mainly control this ability via direct innervation of muscles, allowing them to seamlessly blend in with a variety of environments.16, 20 This ability is potentiated by several classes of dermal organs; the chromatophores, which act like expandable color filters, the iridophores, which are tunable Bragg stack reflectors, and, the leucophores, which reflect diffuse white light, although .20 These organs are layered within the dermis (Figure 1.3), so that the chromatophores are nearest to the surface of the skin, with the iridophores and the leucophores beneath them.13, 20 Iridophores and leucophores produce colors through structural coloration, while chromatophores are thought to be primarily pigmentary organs.15-16

1.3 Iridophores

Iridophores are named after the optical effect that they exhibit, iridescence; a well-known consequence of structural coloration which results in angle-dependent reflection of light.21 One possible use for these reflectors is communication between individuals in a school. Due to the iridescent nature of iridophores, reflectance patterns are indicative of specific movements, and individuals may be able to interpret these patterns to coordinate with the school.15 While not definitive, it has also been suggested that, because iridescence produces polarized light, this could be used as an additional form of communication that is specific to species able to perceive differences in polarization.22 In addition to these uses, iridophores contribute to dynamic

13 coloration for camouflage and signaling by interacting with the overlying chromatophores to produce a wide variety of colors that cannot be achieved by chromatophores alone.23

Figure 1.3. Cross-sectional transmission electron microscope (TEM) image of Octopus dofleini dermis under 9000x magnification. An expanded chromatophore (CO) is visible near the top of the image. Leucophores (L) with labeled leucosomes (LS) are shown below the chromatophore. Iridosomes (RC) are shown with parallel platelets clearly defined.20 Image reproduced from Ref. 20.

14 The iridescent effect of iridophores is made possible by alternating platelets of protein, also known as iridosomes, and extracellular medium (Figure 1.3). The difference in refractive index between the platelets and the medium gives rise to specular reflectance, and the regular spacing between platelets causes constructive and destructive interference (Figure 1.4a), where some wavelengths are amplified while others are decreased according to the relationship shown in equation 1.1.21, 24

푚휆 = 2(푛푎푑푎 cos 휃푎 + 푛푏푑푏 cos 휃푏) (Eq. 1.1)

Using this multilayer interference model, where m = 1 for first order reflection, solving for

λ gives the peak reflected wavelength, the wavelength that is most enhanced by constructive interference (Figure 1.4b). The peak reflected wavelength depends on the refractive indices of the materials (na, nb) and the thicknesses of the layers (da, db). The final components in his model are

θa and θb, the angles of refraction in the corresponding materials, which give rise to the angle- dependent iridescence observed in iridophores as well as other natural multilayer interference systems, such as the metallic-looking elytra of Japanese jewel beetles,25 the iridescent cells of neon tetra,26 and the bodies of copepods.27

15 Figure 1.4. Effects of multilayer interference. a) Diagrammatic representation of a multilayer system. A and B are two materials, each with a specific thickness, d, and refractive index, n. b) calculated reflectance for an ideal multilayer reflector with varying number of layers. The refractive indices are set to 1.6 and 1.55, and the distances are set so that dana=dbnb=125 nm, and the chosen angle of incidence is 90º. These parameters simplify eq. 1 so that for a first order reflection (m=1), λ=500 nm.24 Reproduced from Ref. 24.

In the specific case of cephalopod iridophores, this effect arises from a combination of reflectin, a high refractive index structural protein, and cellular structure.28 Reflectin is unique to cephalopods as a natural, self-assembling protein that can simultaneously function as a reconfigurable multilayer reflector, also known as a Bragg reflector.29-30 While these platelets were observed as early as 1909,31 their composition remained unknown until 2004, when reflectin from

Euprymna scolopes, the Hawaiian bobtail squid, was isolated, characterized, and sequenced.32

While the size and number vary between isoforms, reflectin consists of several repeating subdomains connected by less conserved linking regions.30, 32-35 These conserved regions consist of both hydrophilic and aromatic residues in relatively high abundance, contributing to reflectin’s ability to aggregate, and the addition of aromatic compounds to reflectin has been shown to enhance this aggregation, resulting in higher-order assemblies, presumably based on π-π stacking interactions.29-30, 34 Reflectin, in this condensed, aggregated form, produces the high refractive index layers in the Bragg reflector, while the lower refractive index gaps between the reflectin

16 platelets comprises extracellular space created by the invagination of the cellular membrane around the reflectin platelets, as seen in Figure 5.28

To fully characterize the Bragg stack reflector, the refractive indices of both the reflectin platelets and the extracellular media must be known, along with their respective thicknesses.

Lamellae thickness can be measured with microscopy techniques, such as transition electron microscopy, and the extracellular media can be easily isolated, but refractive index of native reflectin has proven somewhat more complicated.35-36 The refractive index of dehydrated reflectin has been measured as 1.59, which is uncharacteristically high for proteins, but this does not reflect the native state of reflectin in iridophores, which are at least partially hydrated.35 A more accurate method is to calculate refractive index by measuring reflectance of the intact iridophore and fitting it to the multilayer interference model, given that the other variables are known, which gives a refractive index of 1.405 for live cells, and a slightly higher value of 1.413 for chemically fixed cells.37 The refractive index has been confirmed by replacing the extracellular fluid with fluids of progressively higher refractive index, until the reflectance reaches a minimum, providing an effective refractive index of n=1.44.36

In some octopus and cuttlefish, iridophores appear to be static structures, and while the reflected color depends on the angle of reflection, this effect does not change over time.38 These animals generally use chromatophores to modulate the reflections of iridophores for selective communications and dynamic camouflage.16 On the other hand, some species of squid have exhibited active control of iridophore iridescence.39-40 In early observations, acetylcholine was shown to induce iridescence in concert with a reduction of platelet thickness and increase in space between platelets.41 More recent studies have elaborated on this mechanism, showing that acetylcholine application begins a signaling cascade that leads to specific phosphorylation of the

17 reflectin, which drives condensation of the reflectin plates and expulsion of water from the cell, as shown in Figure 5.28, 42 Whether changes in iridescence are due to a change in the refractive index of reflectin or the change in thickness or spacing of reflectin platelets, this change occurs relatively slowly, on the order of minutes, compared to the muscular control of chromatophores, which can occur in under a second.

Figure 1.5. Illustration of the mechanism of neurologically controlled tuning of iridophores. Acetylcholine (ACh) interacts with a type G protein-coupled receptor (mAChR), which initiates a signaling cascade. This causes calcium (Ca2+) release from the endoplasmic reticulum, which acts as a secondary messenger that binds calmodulin (CaM), activating protein kinases (PK) and phosphatases (PP). This results in the phosphorylation and dephosphorylation of specific reflectins followed by the expulsion of water, causing a change in thickness and refractive index of the reflectin platelets and altering the reflective properties of the Bragg stack reflector.28 Reproduced from Ref. 28.

18 1.4 Leucophores Leucophores do not directly contribute to dynamic coloration but provide a static background of diffuse white light independent of viewing angle.15, 20, 43-44 Previously, leucophores were considered to consist of a specialized cell, the leucocyte, that contains a high concentration of spherical particles known as leucosomes (Figure 1.3), while more recent studies have also shown the presence of iridocytes within leucophores which may also contribute to reflectance.20,

38 Leucosomes range in diameter from 500 nm to 4 um and have a refractive index of 1.51.43-44

The difference in refractive index between the leucosomes and the cytoplasm results in broadband scattering, as has been confirmed by Mie theory modeling.43 This is especially apparent in “zebra” displays, like in Figure 1.2, where contrasting bands of light and dark are created by either dilation or contraction of the darker chromatophores over the reflective leucophores.15, 19 While the white leucophore-based stripes cannot be directly controlled, the extent of chromatophore expansion can be modulated to either enhance or weaken contrast to achieve specific camouflage or signaling motifs.43

In general, leucophores are absent from squid, although they are commonly found in cuttlefish and octopus. One notable exception to this is found in D. opalescens. The female of this species of squid features switchable leucophores which reflect bright white light (Figure 1.6).44

Like adaptive iridophores, this process can be initiated by the injection of acetylcholine, and proteomic analysis shows the presence of reflectin, suggesting that the same process of condensation that produces iridescence in tunable iridophores is responsible for the formation of reflectin-based particles in adaptive leucophores.42, 44 This finding, along with the discovery that static leucophores also contain reflectin,43 suggests that reflectin is the basis for structural coloration not only in iridophores but also in leucophores.

19

Figure 1.6. Tunable leucophores in female Doryteuthis opalescens, stimulated by acetylcholine (ACh). A) Reflectance spectra as a function of time after ACh injection. The reflection of white light increases over 200 seconds. B) Images of the D. opalescens mantle after ACh stimulation. The red cross marks the injection sight.44 Reproduced from Ref. 44.

20 1.5 Chromatophores

Chromatophores starkly contrast with the reflective cells, leucophores and iridophores, in terms of structure, mechanism of coloration, and physiological control. While the reflective cells show physiological control in some cases, chromatophores are the main driver of physiological color change in cephalopods, making it possible for these animals to quickly and accurately alter their appearance for the aforementioned purposes of camouflage and communication.

While iridophores and leucophores are well described as aggregations of specialized cells, chromatophores comprise entire organs in cephalopods. Cloney and Brocco, in 1983, described chromatophores as consisting of five different cell types (Figure 1.7).20 Most obviously, there is a central pigment cell, also referred to as the chromatocyte. The source of color in the chromatocyte are pigmented granules within a central granule-containing sacculus. Radially distributed around the chromatocyte are muscle fibers with associated axons and glial cells. These muscles are directly innervated by the animal allowing for neuromuscular control, such that when they contract, the chromatocyte expands. Finally, so-called sheath cells are found in layers covering the rest of the cell types. It had been previously thought that these sheath cells applied a contractive force on the expanded chromatocyte,31 but later work suggests that the elastic nature of chromatophores comes from the sacculus, now referred to as the cytoelastic sacculus, within the chromatocyte itself, leaving the precise purpose of these cells undetermined.16, 20, 45 The cytoelastic sacculus, while not fully characterized, is composed of isotopically oriented filaments, possibly giving rise to an elasticity described as similar to nylon20, 45 The sacculus is connected to the muscle fibers via structures that Cloney and Florey, in 1968, termed haptosomes, supporting the idea that the expansive work performed by the contraction of the muscles is stored in within the sacculus directly as elastic energy.20

21

Figure 1.7. Drawing of the cephalopod chromatophore modified from Cloney and Florey45 by Richard E. Young. Sheath cells are omitted for clarity. Radial muscle, axon, and glial cells surround a central pigment cell. The central cell has an obvious nucleus, as well as a cytoelastic sacculus containing pigment granules. Mitochondria are highly concentrated in the muscles, but mostly absent from the pigment cell, reinforcing the idea that chromatophore expansion is caused my muscular contraction, while relaxation is a result of elasticity. This image is licensed under a Creative Commons Attribution 3.0 NonCommercial License and can be found at www.tolweb.org. Rather than controlling each chromatophore individually, like a computer controlling pixels, cephalopods appear to use a hierarchical control system, where chromatophores are controlled in groups to produce a limited number of global patterns.46 Nevertheless, neuromuscular control means that chromatophores can expand and contract very quickly compared to the examples of physiologically controlled iridophores and leucophores. Actuation in the chromatophore occurs in less than a second, as opposed to the minutes it takes for acetylcholine release to trigger reflectin aggregation in the iridophores.39, 46

The color seen in chromatophores is due to the presence of pigmented granules within the chromatocyte. The range of colors displayed by chromatophores is due to different pigmentation in the granules, and the variety of chromatophore colors varies by species. D. pealeii and S.

22 officinalis, are known to feature red, yellow, and brown chromatophores, while Octopus vulgaris exhibits orange and black chromatophores in addition to the red, yellow, and brown varieties.16, 47-

48 While melanin has been suggested as a possible pigment in these cells, this has not been well supported by evidence.16 The development of chromatophores, in which dark chromatophores begin as pale yellow and continuously darken to become red, then brown,46, 48 is inconsistent with melanin being the primary contributor to color in the pigment granules, with the exception, perhaps, of the genus-specific black chromatophores in O. vulgaris.

In 1976 a study by Van Den Branden and Decleir showed that three different pigments can be selectively extracted from the skin of the cuttlefish, with the different pigments possibly corresponding to the red, yellow, and brown chromatophores.47 They removed dorsal skin from S. officinalis and performed three consecutive extractions, first with tris buffer at pH 7.2, then with

0.1 M HCl in methanol, then with formic acid, affording three pigments labeled A, B, and C. It was observed that the extracted pigments changed color upon reduction, oxidation, and exposure to sulfuric acid (Table 1.1) consistent with the previous suggestion that chromatophore pigments were ommochromes.49-50

Table 1.1. Properties of pigments extracted from S. officinalis skin, according to Branden and Decleir.47 Pigment Pigment Color In Extraction Reduced Oxidized Halochrome Fluid (+Ascorbic Acid) (+H2O2) (+ H2SO4) A Yellow Pink purple Yellow Light purple B Brown red Red purple Yellow Brown purple C Violet purple Violet purple Yellow Violet purple

23 All three of the pigments exhibited a yellow color when oxidized, while under reducing conditions they appeared various shades of purple. Van Den Branden and Decleir also performed a solubility study on the extracted pigments, showing that the all of the pigments exhibited no or very poor solubility in pure alcohols and acetone but were strongly soluble formic acid, 0.1 M HCl in methanol, and 20% potassium hydroxide solution. Additionally, pigments A and B were found to be soluble in neutral water, and acidic buffers. This led to the hypothesis that the three chromatophores in S. officinalis is due to three separate ommochrome pigments, but the precise identity of those pigments remained unknown.47

In an effort to better understand the composition of these complex organs, an analytical characterization of the chromatophores inspired a proteomic exploration of the pigment granules.13

Individual brown chromatocytes were isolated from S. officinalis using laser-capture microdissection. These chromatocytes were lysed, and the resulting proteins were separated using sodium disulfate polyacrylamide gel electrophoresis (SDS-PAGE), tryptically digested, and sequenced via liquid chromatography coupled to tandem mass spectrometry (LCMS/MS). To elucidate which proteins were specifically associated with the granules, the granules were isolated via centrifugation and treated with 0 M, 0.1 M, and 0.2 M sodium hydroxide before being sequenced by LCMS/MS. In order to determine which proteins were released in accordance with granule denaturation, proteins were categorized using self-organizing map analysis.13 This analysis revealed the presence of not only reflectin, just like iridophores and leucophores, but also lens crystallin proteins, specifically Ω-crystallin, associated with the granules.13 This finding was significant because Ω-crystallin had previously been identified in invertebrate eyes, but not cephalopod skin.51-52 And while the exact roles of the proteins remained unknown, the authors suggested that small molecule pigments interact with protein nanostructures to enhance coloration.

24 In order to better understand the mechanisms of chromatophore coloration, we first performed an in-depth literature review of the suggested components; ommochromes, crystallins, and reflectin. By looking at these likely components and their properties in other systems, we can inform our studies on pigment granule composition and better interpret their functions.

1.6 Ommochromes and Xanthommatin

Ommochromes (from the Greek for “eye” and “color”)53 are a class of pigments, first discovered in the eyes of insects.49 These pigments are tryptophan metabolites with poor solubility in most common solvents, including water, alcohols, and hexane.49 Much of the early work on ommochromes was performed in the 1940s by Erich Becker at the Kaiser Wilhelm Institute for

Biology, who divided ommochromes into two categories, ommins and ommatins, based on their physical and chemical properties: ommochromes with lower molecular weights were found to be unstable in alkali solutions and were classified as ommatins, while higher molecular weight, alkali stable ommochromes were classified as ommins.54 While both ommatins and ommins appear yellow under oxidizing conditions, their reduced forms differ, with ommatins generally taking on a red color, while ommins are described as violet.49 Soon afterwards, in the 1950s, xanthommatin became the first ommatin to be to be synthesized.49, 55-56 A variety of ommatins have been discovered since then, many of which are formed by modifications of xanthommatin, like rhodommatin, formed by glycosylation of xanthommatin, and ommatin-D, the product of xanthommatin sulfation. While ommins are still phenoxazinone-based products of tryptophan metabolism, these pigments have been shown to include sulfur, which radiolabeling experiments have shown is incorporated from methionine and cysteine in the biosynthesis.49 Because previous observations regarding chromatophore pigments are consistent with xanthommatin or an

25 xanthommatin derivatives47, shown in Figure 1.8, we researched the presence of xanthommatin throughout natural systems to determine how it is synthesized and how it contributes to coloration.

Figure 1.8. Examples of Ommochrome pigments derived from xanthommatin. Ommatins are all shown in reduced form.49, 57, 58 Since the identification of xanthommatin, it has been found as a pigment in a wide variety of insects, as well as in spiders and cephalopods.47, 49, 59-60 In addition, it has been implicated as a possible product of photooxidation in human lenses.61-62 The main structure of xanthommatin, and the other ommatins, is the pyridino-phenoxazine moiety.63 It is this conjugated structure that gives rise to xanthommatin’s ability to absorb visible light, as well as its insolubility and tendency to aggregate.49 Upon oxidation or reduction, xanthommatin can change color, a property known as electrochromism, allowing it to perform as either a red or a yellow pigment.64 These properties, combined with its high refractive index,65 make it an effective pigment as well as a metabolite with chemical functionality.

xanthommatin synthesis has several possible purposes within insects. First, as a pigment, it can be used for communication or as a camouflage. For example, some male dragonflies use the

26 electrochromic properties of xanthommatin to change color from yellow to red upon sexual maturation, which acts as an identifying signal to both possible female mates and competing males.64 On the other hand, the crab spider uses xanthommatin for camouflage, as it reversibly changes from white to yellow by producing or catabolizing xanthommatin, in order to match the flowers on which it perches, waiting for prey.59 Another, possibly more widespread, purpose to the pigment is as a screening pigment. Xanthommatin in insect eyes does not only act to impart coloration to the organism – it also protects the especially sensitive optical organs from ultraviolet

(UV) light by absorbing the damaging radiation and as acting as an antioxidant or radical scavenger.49, 66 A third explanation for the prevalence of ommochromes in insects is simply a lack of alternate tryptophan catabolic pathways. In mammals, for example, most excess tryptophan is converted into CO2 and acetate via 3-hydroxy-anthranilic acid in the glutarate pathway, as shown in the red steps of Figure 9.49 Insects, lacking this pathway, face a build-up of tryptophan and other potentially toxic intermediates, analogous to glutaric acidemia type 1 in humans which can result in brain damage and premature death.67 Xanthommatin in this case acts as a metabolite sink, since it can be isolated in metabolically inactive granules.68

Xanthommatin biosynthesis begins with tryptophan and proceeds via the kynurenine metabolic pathway (Figure 1.9). Tryptophan is an essential amino acid in animals, meaning that it must be acquired through diet or from symbiotic bacteria and fungi.69 Even though the amino acid is toxic, it is versatile as both a protein building block and as a starting material for various metabolites, including serotonin and melatonin as well as ommochromes and their precursors, due to its indole functional group.49, 70 The first step in the kynurenine pathway of ommochrome biosynthesis is the enzymatic cleavage of the pyrrole portion of tryptophan’s indole group by tryptophan 2,3-dioxygenase or the less specific indoleamine 2,3-dioxygenase.71-72 These enzymes

27 cleave the pyrrolic double bond via oxidative addition of molecular oxygen, catalyzed by the protein’s prosthetic heme group, producing N-formylkynurenine.73

N-formylkynurenine is hydrolyzed to form kynurenine and formic acid.49 This reaction can occur spontaneously, or it can be catalyzed by kynurenine formamidase.49, 71 This process has been studied in Drosophila melanogaster, where crystal structures revealed a binding site where the formyl carbon can bind to a serine residue, making an intermediate oxyanion which stabilizes the complex during hydrolysis.74 The resulting kynurenine is then modified by kynurenine monooxygenase, which adds a hydroxyl group to kynurenine, producing 3-hydroxykynurenine.49

Both kynurenine and 3-hydroxykynurenine are known to produce reactive oxygen species when illuminated by UV light, causing oxidative stress in sensitive organs, though, paradoxically, kynurenine has also been shown to have a neuroprotective effect.62, 66, 70, 75

28

Figure 1.9. Biological synthesis of Xanthommatin. The first step, in which tryptophan is converted to N-formyl kynurenine, is catalyzed by indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3- dioxygenase (TDO). Kynurenine formamidase (KFase) removes the formyl group to produce kynurenine and formate. Kynurenine 3-monoxygenase (KMO) adds the phenyl group to produce 3-hydroxykynurenine (3-OHK), which then forms dihydroxanthommatin. Along this pathway, though, there are several competing reactions. For example, kynurenine aminotransferase (KAT) can convert kynurenine to kynurenic acid or 3-OHK to xanthurenic acid. Kynureninase, found in humans, but not in insects, convert kynurenine and 3-OHK to anthranilic acid and 3- hydroxyanthranillic acid.49, 67, 76 The production of 3-hydroxykynurenine from tryptophan is important not only in ommochrome synthesis but also in the glutarate and NAD pathway to produce acetate and nicotinamide adenine dinucleotide, respectively.67 Additionally, 3-OHK is widely distributed through nature, as supported by the ubiquitous presence of KMO across unicellular and multicellular life, shown in Figure 1.10a. Figure 1.10b shows the distribution among animals in more detail, confirming that this enzyme has been reported previously in a wide variety of insects and arachnids, as well as in a cephalopod species, O. bimaculoides, but KMO is also

29 present in a wide variety of vertebrates. In contrast, the distribution of xanthommatin in nature appears to be limited to insects, arachnids, and cephalopods, suggesting that, although 3-OHK is ubiquitous, the final step to produce xanthommatin is relatively rare, either due to a missing enzyme or competing metabolic pathways .49, 76 Perhaps because of this, there have only been a few biochemical studies of the pigment since the 1980s, with several papers in the last decade finally exploring the final steps of xanthommatin synthesis.76-79

The final step in xanthommatin synthesis is the dimerization of 3-hydroxykynurenine.

This can occur enzymatically or via autooxidation.62, 80 For example, autooxidation of 3- hydroxykynurenine can occur in human lenses and might be a factor in the development of cataracts.61-62 In insects, xanthommatin synthesis has been shown to occur by nonenzymatic oxidation, as in the mosquito, Aedes aegypti, while the presence of phenoxazinone synthases has been long suggested in D. melanogaster, but has yet to be isolated and characterized.81 In fact, recent studies have identified cardinal, a gene that codes for peroxidase, not phenoxazinone synthetase, as a key gene in the conversion of 3-hydroxykynurenine to xanthommatin in Diptera, the order of insects including flies and mosquitos, as well as cardinal analogs in two other insect orders: Lepidoptera, specifically in silkworms, and Hemiptera, in a delphacid planthopper.78-79 A different enzyme, tyrosinase, has been shown, in vitro, to have phenoxazinone synthetase activity.82 Still, a phenoxazinone synthase for the catalysis of 3-OHK dimerization is likely required in some animals in order to compete with the other pathways shown in Figure 1.9.

30

Figure 1.10. Phylogenetic organization of kynurenine monooxygenase distribution in nature. The UniProt database was searched for the protein “kynurenine monooxygenase.”83 The 3,535 results were exported and condensed into 921 unique genuses which were plotted using the iTOL online tool.84 a) Presence of the enzyme kynurenine 3-monooxygenase (KMO) throughout bacteria (green), fungi (blue), and metazoa (purple), but almost completely absent from plants. b) Distribution of the enzyme throughout the animal kingdom (Metazoa). The majority of animal species with KMO are either protostomes (red) or deuterostomes (dark blue). These two evolutionary clades are further split into large, recognizable classes or orders: Sauria (green), Mammalia (purple), Actinopterygii (light blue), Insecta (orange), Arachnida (brown), and Nematoda (light green). KMO was only reported in one cephalopod species, Octopus bimaculoides.

1.7 Pigment Granules in Insects

Xanthommatin is abundant in arthropod eye and skin cells.49 It has also been detected in cephalopod chromatophores.60 In most cases, the pigment is found within pigment granules, spherical structures consisting of dihydroxanthommatin, the reduced form of xanthommatin, and

“matrix proteins.”13, 59, 85-87 In insects and spiders, each pigment granule exists within a vesicle,

31 while cephalopod chromatophore cells contain a specialized cytoelastic sacculus which houses a network of many pigment granules.13, 87

While there is a paucity of information on granule genesis in cephalopods, studies on insect and spider pigment granules can provide some context as to the processes involved (summarized in Figure 1.11). In these arthropods, there is evidence that pigmentless progranules are produced by the rough endoplasmic reticulum and exported in vesicles.59 3OHK synthesis, as described above, occurs outside of the progranule, likely in the cytosol, and the product is transported into the pigment granule via ATP-binding cassette (ABC) transporters consisting of two ATP-binding domains and two transmembrane domains.88-90 While the mechanism is not fully elucidated, it is thought that the transmembrane domains selectively bind 3OHK and conformational changes, induced by the binding or hydrolysis of ATP, carry it through the membrane into the vesicle, where it undergoes oxidative or enzymatic condensation into xanthommatin or dihydroxanthommatin.49,

85, 88, 91

In both insects and spiders, pigment granules comprise both these pigments and various proteins. Early insect studies suggested that dihydroxanthommatin condenses around the phenoxazinone synthetase in a self-inhibiting manner.86 Other studies, focusing on butterflies and moths, show the presence of ommochrome-binding proteins in the granules.68, 92 Still, the exact mechanism of granule formation in these species remains largely unknown.

32

Figure 1.11. Suggested model of ommochromosome formation in spiders. Tryptophan (Trp) is converted to N-formylkynurenase (FKyn) by tryptophan 2,3-dioxygenase (TDO) within the cell, followed by conversion to kynurenine (Kyn), possibly catalyzed by kynurenine formamidase (KFase). Kyn is oxidized by kynurenine monooxygenase (KMO) at the mitochondria to form 3OHKyn (3-hydroxykynurenine). ABS transporters carry 3OHKyn into the ommochromasome, where phenoxazinone synthase (PHS) or Cardinal may catalyze production of ommatins. This model is based on the crab spider which can change color by producing or catabolizing ommochromes, allowing microscopy to expose granules in various stages of growth. Reproduced from Ref. 76.59, 76, 87

1.8 Reflectin and Crystallin

Previous research by Deravi et al. described the presence of proteins in the brown chromatophores of S. officinalis, associated with pigment granules, consistent with these arthropod analogs, but featuring cephalopod specific proteins.13 Among these proteins, crystallins and reflectin show promise in terms of contributing to the optical properties of chromatophores. Reflectins have been identified in iridophores and in leucophores, in which they

33 contribute to multilayer interference reflectors and broadband light scattering.32, 38, 43 Crystallins, on the other hand are normally found in the lens of eyes.52, 93 While reflectins may impart structural coloration in specific configurations, lens proteins evolve specifically to have high refractive index without scattering or absorbing light, as these behaviors negatively impact vision.

Although crystallins are found in eyes throughout the animal kingdom, they vary in structure and composition.52 For example, two of the crystallins found in vertebrate eyes, α- and

β-crystallin, are very different from each other. α-crystallin appears to have evolved from small heat shock proteins and forms large aggregates weighing an average of 800 kDa, while β- crystallin shows homology with γ-crystallin and forms dimers or oligomers ranging from 50-200 kDa.94-95 Even though lens crystallins vary in structure, they do share common properties. They must be transparent and exhibit a high refractive index in order to function in the lens, and they must be remain soluble, since protein aggregation causes scattering which negatively affects vision in the form of cataracts.96

The main crystallin isoforms specific to cephalopod lens are S-crystallin and Ω-crystallin, while a third, less abundant lens protein, O-crystallin, has only been reported in octopus and has not been fully characterized.52, 93 S-crystallin has been discovered in cuttlefish, squid, and octopus lens, and is also reported to also exist within cuttlefish pigment granules, showing that this protein is widely distributed throughout the coleoid cephalopods.13, 52, 96 Ω-crystallin was first reported octopus lens, but has also been found in the photoluminescent organ of E. scolopes, the Hawaiian bobtail squid.51-52, 97 Since both crystallins are found in cephalopod skin as well as lens, we will describe both in detail.

34 S-crystallin is related to glutathione S-transferase but with diminished or absent enzymatic activity.52, 98 While this protein satisfies the requirements of a crystallin by increasing the refractive index of the lens, it also exhibits some unique features. S-crystallin shows some affinity for glutathione, which is hypothesized to reduce aggregation that leads to lens clouding.96 S-crystallin also features a surprising variety of genetic variants. A total of 53 S- crystallin transcripts were found via RNA sequencing of D. pealeii lens tissue, with most of this variation due to the insertion of a variable length loop.96, 99 It was also observed that the refractive index of the lens changed in a gradient, where material taken from the edge of the lens has a refractive index similar to water, while material at the center of the lens approaches the limit for dry protein, 1.6.100 This change of refractive index over lens radius matches the ideal relationship to minimize spherical aberration in a curved lens, made possible by interactions between S-crystallin which lead to the assembly of a so-called “patchy colloid,” which is assisted in part by the polydispersity of S-crystallin monomers.100

Ω-crystallin is a tetrameric protein highly homologous to aldehyde dehydrogenase

(ALDH).52, 97, 101 ALDH is unique in that it occurs as a crystallin in both invertebrates, as Ω- crystallin in cephalopods and scallops, and in a vertebrate, as η-crystallin in the elephant shrew.52, 102 Even though these crystallins are closely related to ALDH, they are enzymatically inactive, suggesting that they have been specialized for expression in the lens.97, 101

Unlike the S- and Ω- isoforms of crystallins, reflectins are not homologous to any known protein.30, 32 Instead of being recruited from existing enzymes, a reflectin precursor was likely introduced into cephalopods via horizontal gene transfer from a symbiotic bacterium. This is supported by the presence of a 24 base pair gene fragment that is found in both cephalopod reflectin and two transposase genes in Vibrio fishcheri¸ a bacterium known to live symbiotically

35 with squid.30 This gene fragment codes for the “protopeptide,” with the amino acid sequence

YMDMSGYQ. In reflectin, this sequence is repeated several times (Figure 1.12a), which indicates that the protopeptide gene underwent gene duplication sometime after horizontal gene transfer from the V. fishcheri. The conserved, repeated nature of this protopeptide suggests that it may be play an integral role in the structure or function of reflectin. The effect is similar to that of a block-copolymer in reflectin, where the protopeptide and its flanking regions contain a relatively large amount of the aromatic amino acids, tyrosine, phenylalanine, and tryptophan, leading to π-π stacking interactions.30, 34-35 These π-stacking regions, interspersed with charged residues, especially aspartic and glutamic acid, lead to self-assembly of reflectin particles, films, and larger structures.

Figure 1.12. a) Diagrammatic representation of reflectin based on a reflectin gene from cuttlefish (SoRef2). Reflectin consists of four connected domains (D1-D4), each of which have a core region (Core) consisting of three repetitions of the protopeptide. Below, sequences of these four core regions are shown, along with the non-conserved N-terminal domain (ND). A tag cloud, labeled consensus, shows relative frequencies of each amino acid in each position of the protopeptide. b) The percent abundance of each amino acid coded for within the SoRef2 gene. The four aromatic amino acids are indicated with their respective structures.30 Reproduced from Ref. 30.

36 Beyond the optical and structural properties of reflectin, there is another property which may impact its function not only within pigment granules but perhaps in iridophores and leucophores as well. An in vitro study using bacterially expressed reflectin showed that reflectin films act as proton conductors in addition to their optical function as Bragg stack reflectors.33 A prepared reflectin film shows conductance only when hydrated, consistent with proton conductance via the Grotthuss mechanism, in which protons jump between water molecules.33

The measured conductivity of reflectin, ~1x10-4 S cm-1 is almost three orders of magnitude lower than that of an artificial proton conductor such as Nafion, measured at 7.8x10-2 S cm-1 , but it is quite high compared to other protein films.103 The authors suggest that this is made possible due to the uncommon amino acid composition of reflectin, which includes a significant abundance of both aromatic amino acids and charged amino acids, shown in Figure 1.12b.30, 32, 34 While the aromatic amino acids are implicated in the assembly of reflectin platelets, the charged amino acids may create hydrophilic water channels through which protons can be conducted, as shown by the loss of conductance when aspartic acid and glutamic acid are mutated to alanine. Even though proton conductance of reflectin has, thus far, only been shown to occur in vitro, it does suggest the possibility that reflectin may play an active role in neurological signaling, rather than only act as a tunable thin film.

1.9 Cephalopod Inspired Materials

Currently, we have a general understanding of some basic mechanisms underlying the ability of cephalopods to dynamically alter their coloration. It is important to improve on this understanding in order to inform the design of new technologies. Cephalopod biology has already inspired a variety of technology, including dynamic infrared (IR) camouflage, visible displays, and functional soft materials. These technologies recreate the effect of various structures within the

37 cephalopod skin: the structural coloration produced by iridophores, the areal expansion of chromatophores, and the texture modifying ability of dermal hydrostats known as papillae.

IR camouflage has been a focus of Alon Gorodetsky at the University of California Irvine.

This technology would be advantageous in a military context, as IR “night” vision relies on the differential reflection of IR light to identify objects and people.104-106 It had previously been determined that the protein platelets in iridophores are composed of reflectin, and that the optical properties of iridophores were created due to alternating layers of high refractive index reflectin and lower refractive index cytoplasm each with well-defined thicknesses.21, 32 Inspired by the tunable reflection of iridophores, Gorodetsky produced thin films from microbially expressed reflectin.105-106 While this doesn’t exactly replicate the multilayer interference caused by iridophores, thin film interference caused by a single layer of reflectin is similar in mechanism, and the peak reflected wavelength still depends on the thickness of the reflectin film, as described using equation 1.2.21 By increasing the thickness of the reflectin film, the peak reflected wavelength can be shifted into the IR region, making these films tunable IR reflectors.

푚휆 = 4푛푑 cos( 휃)(Eq. 1.2)

While these films could be produced from materials other than reflectin, reflectin provides additional useful properties. Reflectin films have refractive index of 1.59, similar to the native protein.23, 35 The self-assembly of reflectin makes the films mechanically robust and temperature resistant, making them amenable to common processing techniques.33 Finally, the thickness of the film can be adjusted by controlling hydration, just like iridophores, providing one method of tuning iridescence, Additionally, it was found that acetic acid vapor was causes a larger increase in thickness and therefor a larger red-shift of the reflected light.106 In this way, the reflected light can be switched between visible and IR, so that the film can be used as an active IR camouflage.

38 An alternative method of tuning the reflectance profile involves physically stretching the reflectin film, removing the need to control film hydration.105 These “IR invisibility stickers” consist of a reflectin film on a stretchable tape. As the tape is stretched, the reflectin stretches as well, and the deformation causes the reflectin film to become thinner. As expected according to equation 2, the thinner film reflects light at shorter wavelengths. Gorodetsky and coworkers produced one such device with a reflectin film thickness of 160 nm. In an unstrained state, the peak reflection occurred at approximately 980 nm, but when strained, the thickness of the film is reduced to about 120 nm with a concomitant blue shift of the peak reflectance to 705 nm, in accordance with thin film interference theory. In both cases, the reflectin film can be adjusted to reflect more or less IR light by increasing or decreasing film thickness. Applied to, for example, clothing, this would allow a person to avoid detection by IR cameras by selectively reflecting IR light to match the surrounding environment (shown in Figure 1.13a).

39

Figure 1.13. Examples of cephalopod inspired technology. a) Top: digital picture of fluorinated ethylene propylene tape, the reflectin-coated tape, and a leaf placed on top of cloth with camouflage pattern. Bottom: The same items imaged with an infrared camera, under infrared illumination, showing how reflectin alters the optical properties of the tape to more closely match the leaf.105 Reproduced from Ref. 105. b) Artificial chromatophores based on dielectric elastomers, shown in an actuation cycle. Black and white images show which elastomers are expanding or contracting.107 Reproduced from Ref. 107. c and d) Three dimensional inflatable elastomers based on cephalopod papillae.108 Reproduced from Ref. 108. Some visual displays have already been developed using cephalopod chromatophores as inspiration. Cephalopod skin acts like a reflective display where the expansion of a chromatophore is similar to a pixel turning on. Reflective displays are generally more energy efficient than other types of displays because they reflect ambient light, while transmissive displays require a backlight to be seen, especially in brightly lit environments.109 One method of creating reflective displays

40 based on cephalopod skin, as described by Rossiter and coworkers, involves the use of dielectric elastomers consisting of a polyacrylate film coated with carbon grease electrodes.110 A large electrical potential applied across the dielectric elastomer causes areal expansion in the film, as the difference in voltage causes electrostatic forces between the electrodes.111 Rossiter and coworkers designed an “artificial chromatophore” consisting of three pigmented dielectric elastomers stacked on top of each other, but offset so that they only overlap when actuated.107

Instead of the red, brown, and yellow colors of natural squid chromatophores, these chromatophores were colored red, green, and blue, the primary colors found in pixel displays (as shown in Figure 1.13b). Rossiter predicted that this configuration can produce a wide range of colors though the overlap of any two, or all three, of the pigmented actuators, and that the coordinated action of these chromatophores can impart a global color change, similar to the natural system.107 More recently, Rossiter has published work focused on coordinating the expansion of multiple dielectric elastomers in a network in order to reproduce patterns seen in cephalopod skin.112

Finally, the texture-changing ability of cephalopod skin has inspired the production of soft materials. Cephalopods can alter not only the appearance but also the shape of their skin using organs known as papillae.108, 113 These organs exist in a variety of shapes and sizes and each can be extended or retracted to alter the texture of the cephalopod skin, not dissimilar to how chromatophores work together to produce a global color change within the animal.113 Papilla actuation is possible because they act as muscular hydrostats in which erector muscles provide both contractive force as well as structural support.113 The organization of muscle fibers in papillae inspired Shepard and coworkers to design stretchable membranes that produce pre-determined three-dimensional shapes when inflated (shown in Figure 1.13c and d).108 This works by cutting

41 concentric rings into a non-extensible mesh then encasing the patterned mesh in an elastomer.

When the elastomer is inflated, the patterned mesh provides structure analogous to the erector muscles. In this way, the two-dimensional mesh directly controls the three-dimensional conformation of the inflated elastomer. The authors call this mechanism CCOARSE,

Circumferentially Constrained and Radially Stretched Elastomer, and it allows for the production of flat materials that can be reversibly actuated to create customizable textured surfaces.108

1.10 Dissertation Aims

In order to replicate the quick and accurate camouflage of cephalopods in new technologies, better understanding of the mechanisms underlying chromatophore coloration is necessary. Starting at a molecular level, this means confirming the identity of the pigments in chromatophores and characterizing its optical and electrochemical properties. These pigments are known to condense into granules with diameters of about 500 nm that are suspected to contain pigments as well as proteins; thus, another goal of my work is to elucidate the supramolecular mechanism for granule assembly in the chromatophores. Furthermore, I will discuss new methods of synthesizing the ommochrome pigments involved are required. In order to achieve this, an electrosynthetic technique is applied to the dimerization of 3-OHK in order to produce

Xanthommatin. This replaces the previous synthesis using potassium ferricyanide pioneered by

Butenandt et al. in the 1950s with a more scalable alternative.55

Chapter 2 briefly discusses the methodology used in experiments throughout chapters 3,

4, and 5. Chapter 3 relates the initial chemical evaluation of the composition of chromatophores.

Squid D. pealeii are dissected to isolate pigment granules from their chromatophores. Additionally, pigment is extracted from the granules for independent analysis. The optical and electrochemical properties of the granules and pigment are characterized and compared to synthetic xanthommatin.

42 Mass spectral data confirms the identity of the extracted pigment as xanthommatin. Chapter 4 discusses the proteomic exploration of squid chromatophores. Individual chromatophores were manually isolated from D. pealeii skin and sorted by color. Brown, red, and yellow chromatophores were lysed, digested, and analyzed by liquid chromatography/mass spectrometry.

Detected peptides were searched against an annotated transcriptome from D. pealeii chromatophores, and BLAST was used to identify and categorize the detected proteins.

Microscopy and computational data are included to explain the significance of these findings.

Finally, Chapter 5 details the new electrosynthetic method for the scalable production of xanthommatin. We explore the mechanism behind xanthommatin dimerization from 3-OHK using computational modeling in conjunction with previous studies. This synthesis is the first step in scaling up the synthesis for creating bioinspired devices. Chapter 6 concludes this work, connecting it to previous literature as well as laying out some possible future research.

43 Chapter 2: Overview of Methods

2.1 Animal Collection

Adult squid, D. pealeii, were collected by the Marine Resources Center (MBL, Woods

Hole, USA). Because ethics approval for cephalopods is not required in the United States, the

Institutional Animal Care and Use Committee has no authority for review of such protocols.

However, the authors conducted all experiments with sincere efforts towards ethical care and treatment of these animals, where the number of individuals was minimized and an aquatic veterinarian at the Marine Resources Center of MBL was routinely consulted throughout the season. For our experiments, squid were anesthetized in ethanol and sacrificed.

2.2. Chromatophore Isolation

Decapitated D. pealeii were extirpated of organs and ink sacs. The dermal tissue was manually removed with tweezers and the subdermal chromatophore layer was collected. The chromatophore containing skin layer was collected, homogenized, and purified through a series of centrifugation and washing cycles. The isolated granules were stored in the homogenization buffer containing HEPES (100mM), MgCl2 (10 mM), k-aspartate (50 mM), DL-dithiothretol (1 mM), and tablet protease inhibitor (1 tablet) at 4°C until further use.

2.3 Collection of chromatophore granules

Chromatophore granules were collected in a color independent manner. D. pealeii were dissected and extirpated of organs and ink sacs. The outermost hyaline layer of tissue was manually removed with tweezers and the underlying chromatophore layer was collected in sections. The skin sections were aliquoted into miniature centrifuge tubes with 0.5 mL of collagenase and papain solution. Samples were vortexed, sonicated, and centrifuged. The supernatant was removed, and

44 more enzyme solution was added. This was repeated three times total. After the final centrifugation, the supernatant was removed, and a homogenization solution was added. After vortexing and sonicating the mixture, any large tissue pieces were removed, and the remaining suspension was centrifuged. This step was repeated three times to ensure that the enzyme solution was completely inhibited and any unreacted tissue was removed, leaving behind the water- insoluble granules.13, 60

2.4 Pigment Extraction

0.5% (v/v) hydrochloric acid in methanol solution (HCl-MeOH) was added to the isolated pigment granules. The mixture was vortexed (~1 min) and sonicated (5 min). The solution was centrifuged (5 min at 14,000 rcf) and the pigmented supernatant was collected. The extraction was repeated 3-4 times until no further color was collected.

2.5 Pigment Separation

Thin layer chromatography (TLC) is a separation technique in which the mobile phase is

114 drawn up the stationary phase by capillary action. Instead of retention times, the Rf values, the ratio of the distance travelled by the analyte to that of the solvent, is reported, with this value depending on the relative affinity of the compound for the solvent over the stationary phase.114

While this is generally used as a fast and inexpensive analytical method, it can be scaled up for preparative use. Extracted pigment solution was loaded and separated on preparative normal phase silica TLC plates via a 3:1 (v/v) phenol:water solution. The four separated bands were scraped from the plate and air dried. Each band was further dried over a fritted vacuum filter and washed with chloroform and water. Each band was additionally re-separated and re-extracted to increase purity.

45

2.6 Scanning Electron Microscopy

Scanning electron microscopy (SEM) is used to image structures on the nanometer and micron scale by focusing an electron beam onto conductive surfaces and measuring the secondary electrons ejected from the surface.115 Images of pigment granules pre-and post-extraction were taken using a Tescan Lyra3-GMU Focused Ion Beam-Scanning Electron Microscope.

2.7 Ultraviolet-visible light spectroscopy

Ultraviolet-visible light spectroscopy (UV-Vis) measures the absorbance of light by a substance. This absorbance occurs when light excites a molecule, causing an electron to move from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital

(LUMO).116 In this way, the wavelength at which absorbance occurs provides information on the electronic structure of the molecule. The extent of absorbance is directly proportional to the electronic properties of the analyte, simplified as its molar absorptivity coefficient, ε, as well as its concentration (c), and pathlength of the optical cell used (b), as described in the Beer-Lambert

Law (Equation 2.1).

퐴 = ε푏푐 (2.1)

The absorbance of the pigment granules pre- and post-extraction, extract supernatant, and separated bands was measured using a Cary 60 dual beam spectrophotometer using 0.5% (v/v)

HCl-MeOH solution as a blank solution.

46

2.8 Fluorescence Spectroscopy

Fluorescence is the emission of light following absorbance. In fluorescence spectroscopy, the analyte is excited at a specific wavelength, and the intensity of light emitted at a longer wavelength is recorded. At low concentrations, fluorescence is directly proportional to concentration (c), like absorbance, but it also depends on the intensity of the excitation source (P0), as shown in equation 2.2, where k is a constant.116

퐼 = k푃0푐 (2.2)

Either excitation or emission wavelength can be scanned across a range to produce fluorescence spectra. The fluorescence spectrum of each sample was measured using a Cary Eclipse

Spectrophotometer, by exciting at 390 nm and measuring emission intensity from 450 to 750 nm. Three-dimensional fluorescence spectra were created with a custom script that combined emission spectra of samples excited at wavelengths from 250 nm to 700 nm, in increments of 5 nm, adjusting the emission range to minimize signal from first and second order scattering.

2.9 Mass Spectrometry

Samples extracted from TLC bands were analyzed by LC-MS and LC-MS-MS as described previously.117-118 Briefly, 1 ml aliquot of the digestion mixture was injected into an Dionex

Ultimate 3000 RSLCnano UHPLC system (Dionex Corporation, Sunnyvale, CA), and separated by a 75 mm ´ 25 cm PepMap RSLC column (100 Å, 2 µm) at a flow rate of ~450 nL/min.

Separation was carried out with a gradient program, 5-50% solvent B (80% acetonitrile in 0.1% formic acid) over 30 minutes (solvent A: 0.1% formic acid) in a compartment heated to 37 °C. The eluant was connected directly to a nanoelectrospray ionization source of an LTQ Orbitrap XL mass

47 spectrometer (Thermo Scientific, Waltham, MA). LC-MS data were acquired in the Orbitrap with a MS scan (m/z 150-700, 30K resolution), and MSMS data were acquired both in the linear ion trap by low-energy CID analysis and in the Orbitrap by high-energy c-trap dissociation.

2.10 Isolation of chromatophores for whole cell analysis

Individual chromatophores were isolated by removing sections of dermis from D. pealeii.

Prior to removal of dermis, the upper hyaline and epidermal layer was removed. The chromatophore containing skin layer was then removed from the animal, with careful attention to exclude the iridophore layer below. The tissue sections were immersed overnight in a solution of collagenase (~6 mg ml-1 Clostridium histolyticum) in sea water. Samples were triturated to encourage the breaking up of tissue. After a final trituration, the samples were washed five times with filtered sea water or Tris buffer. Finally, individual chromatophores were plucked out of the solution manually via micropipette, aided by stereomicroscope, and they were separated by color.

Brown and yellow chromatophores were collected from dorsal mantle, red chromatophores were obtained from the ventral dermis.

2.11 Protein purification and analysis using mass spectrometry

The yellow, red or brown chromatophore cells were lysed by sample buffer and sonicated to solubilize proteins. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue G250. In gel electrophoresis, an applied electric potential causes charged molecules to migrate through the gel towards the appropriate electrode. The dodecyl sulfate associates with proteins, giving them an overall negative charge so that they migrate towards the anode. The speed of electrophoretic migration is dependent on protein size, as the protein must travel through pores in the gel. This results in protein separation based on molecular weight.119

48

Figure 2.1. SDS-PAGE gels showing separated proteins from whole chromatophores. All three colors of chromatophore consist of a large number of proteins with a variety of molecular weights. Each lane of different color chromatophore cells were divided into 5 bands, reduced and alkylated, digested in-gel with trypsin. Tryptic peptides were extracted with 50% acetonitrile in

2% formic acid. Volume of the digestion mixture was reduced to 3.5 µL, and 1 µL was injected in a Dinonex Ultimate 3000 RSLC nano UHPLC system (Dionex Corporation, Sunnyvale, CA), and separated by a 75 µm x 25 cm PepMap RSLC column (100 Å, 2 µm) at a flow rate of ~450 nl min-

1. The eluant was connected directly to a nanoelectrospray ionization source of an LTQ Orbitrap

XL mass spectrometer (Thermo Scientific, Waltham, MA). LC-MS data were acquired in an information-dependent acquisition mode, cycling between a MS scan (m/z = 315-2,000) acquired in the Orbitrap, followed by low-energy CID analysis on 3 most intense multiply charged precursors acquired in the linear ion trap.

49 For the granules, pigment, and extracted granules, the samples were denatured and separated by SDS-PAGE. After staining the gel with Coomassie blue G250, visible bands from all three samples were excised for in-gel digestion and MS/MS analysis as above.

Figure 2.2. SDS-PAGE gel showing extracted pigment, extracted granules, and intact granules, each performed in triplicate.

2.12 Sample collection and RNA sequencing of squid chromatophore

Existing RNA sequencing data were accessed from the Short Read Archive (SRA) under accession number SRX817967. Briefly, specimens of D. pealeii were collected by the MBL. Fresh

Tissue was dissected from the chromatophore layer of the skin. RNA was extracted with the

RNAqueous kit (Life Technologies, Carlsbad, CA) and stored at -80°C. RNA libraries were prepared using TruSeq Stranded mRNA Prep Kit (Illumina, San Diego, CA) and sequenced using an Illumina HiSeq 2000 instrument.120

50 2.13 Transcriptome assembly

The RNA sequence data were assembled using the Oyster River Protocol.121 In brief, this assembly procedure began by implementing a read-error correction algorithm.122 Next, Illumina sequencing adapters were removed, as were nucleotides whose PHRED score was less than 5.123

The corrected and trimmed reads were assembled, after which the assembly was evaluated using the software packages BUSCO124 and TransRate.125 The transcriptome was annotated using the software package dammit, which included the production of amino acid sequences via open reading frame identification and subsequent translation. The resultant transcriptome was then used as described below.

2.14 Protein identification using BLAST

The centroided peak lists of the CID spectra were generated by PAVA126 and searched against a protein database translated from the transcriptome of squid D. pealeii chromatophores.

A random-concatenated version of this protein database was also generated to assess false discovery rate of protein identification.127 In addition, the Swiss-Prot protein database was also included to accommodate common contaminations in sample handling. Batch-Tag, a program in the in-house version of the University of California San Francisco Protein Prospector version

5.20.23, was used to identify proteins. A precursor mass tolerance of 15 ppm and a fragment mass tolerance of 0.5 Da were used for protein database search. Protein hits were reported with a Protein

Prospector protein score ≥22, protein discriminant score ≥0.0 and a peptide expectation value

≤0.01.128 This set of protein identification parameters threshold did not return any substantial false positive protein hit from the randomized half of the concatenated database. For each of the identified protein entries, the amino acid sequence was searched against Swiss-prot database

(downloaded January, 2017) using the BLASTP (Basic Local Alignment Search Tool) program

51 for function annotation. The BLASTP search was conducted using default parameters, with the exception of the e- value, which was set to 1e-10.

2.15 Spectral Count Data processing

The spectra count for each of the identified protein entries was recorded. For the whole, red, brown, and yellow chromatophore cells, normalization was based on the assumption that the amount of all proteins constituting chromatophore cells of different colors was similar, though the amount of individual proteins might vary. Therefore, the sum of spectra counts for identified protein entries in each color of chromatophore cells was used to calculate the normalization factor for protein loading, 1, 2.2 and 1.8 for yellow, red and brown respectively. Spectra counts were divided by normalization factors before direct comparison of identified protein entries in yellow, red and brown chromatophore cell samples.

In order to compare relative abundance of different proteins within samples, all spectral counts for each protein were normalized by the number of peptide fragments produced upon trypsin digestion. This was done by dividing each protein by the number of peptides it is expected to produce, then multiplying it by 65.5, the average number of expected peptides across the protein dataset used. These numbers were calculated using the Protein Prospector MS-Digest tool at http://prospector.ucsf.edu and further supported by statistical analyses as described in

Supplemental Notes 1. The mass spectrometry proteomics data that support this study have been deposited to the ProteomeXchange Consortium via the PRIDE129 partner repository with the dataset identifier PXD011975.

52 2.16 Synthesis of Xanthommatin

Xanthommatin was synthesized via the oxidative cyclization of 3-OHK using potassium hexacyanoferrate in a phosphate buffered saline solution (0.1 M PBS, pH= 7.4) according to a previous report.55 An orange product was purified using an OASIS WAX solid phase extraction

(SPE) column, washed with water and eluted using 0.5% (v/v) hydrochloric acid in methanol (HCl-

MeOH). The purified product was then characterized using LC-MS, verifying the purified product was attained.

2.17 Electrosynthesis of Xanthommatin

Electrosynthesis of xanthommatin was performed using a Gamry Interface 1000B potentiostat. For up to 120 minutes, a potential of 0.5 V vs Ag/AgCl was applied to a solution of

10 mg of 3-OHK dissolved in 10 mL of 0.1 M sodium phosphate, adjusted to pH 7.2 with hydrochloric acid in a three-electrode cell. The working electrode was a 4 cm x 1.5 cm block of reticulated vitreous carbon. The counter electrode was a platinum wire placed inside a glass tube with a fritted end. The reference electrode was an Ag/AgCl reference electrode. Following the synthesis, the product solution was purified using an OASIS WAX SPE column and eluted using

HCl-MeOH. A semipreparative high performance liquid chromatography (HPLC) separation was performed with an Agilent 1100 series HPLC system using an Agilent Zorbax 300SB-C18 semi- preparative column (240 mm length x 9.4 mm i.d., 5 um particle size), based on a previously published method for separating xanthommatin from dragonflies.64 The elution gradient was based on a binary solvent system [time in min[% solvent A/% solvent B]: 0 min (95/5); 2 min (95/5); 12 min (40/60); 12 min (10/90); 14 min (10/90); 20 min (5/95)]. Solvent A consisted of 0.1% TFA in water. Solvent B consisted of 0.1% TFA in acetonitrile. The column temperature was set to 30°C,

53 and the flow rate was kept constant at 4.5 mL/min. The eluent absorbance at 254 nm, normalized to absorbance at 360 nm. Fractions were collected manually and rotary evaporated prior to use.

2.18 Characterization of Electrosynthesized Xanthommatin

To evaluate product conversion over time, 50 μL aliquots were removed every 5 minutes for the first 30 minutes of the synthesis, then at 45, 60, 90, and 120 minutes. These aliquots were diluted into 2 mL solutions and characterized by UV/Vis spectrophotometry (UV/Vis) and cyclic voltammetry (CV). CV is a three-electrode technique in which current is measured while an applied voltage is cycled between oxidizing and reducing potentials. The measured current is a combination of faradaic current due to a redox reaction, and capacitive current, and provides data regarding the reversibility and redox potentials of redox reactions. CV was performed using the same equipment as for electrosynthesis, except with a glassy carbon electrode as the working electrode. The applied potential was scanned from -0.4 V to 0.6 V at 100 mV/s. UV/Vis absorption measurements were performed using an Ocean Optics Spectrophotometer.

2.19 Confirmation of Electrosynthesized Xanthommatin Structure

Proton nuclear magnetic resonance (1H NMR) was performed to confirm the product’s structure. Samples were dissolved in 0.6 mL d6-DMSO and with 25 μL of TFA and spectra were collected using a 500 MHz Varian Inova instrument. Data for 1 H NMR is reported as follows: chemical shift (δ ppm), multiplicity (s =singlet, d = doublet, t = triplet, q = quartet, p = pentet, m

=multiplet), coupling constant (Hz), and integration. 1 H NMR (500 MHz, DMSO) δ 8.36(d,

J=4.55 Hz, 3H), 8.05 (m, 1H), 7.83(d, J=3.95 Hz, 2H) , 7.69 (s, 1H) , 6.67 ( s, 1H), 4.46 (d, J=5.20

Hz, 1H), 3.89 ppm (m, 2H) (Figure 2.1) TFA is seen as a broad band centered at 12.8 ppm.

54 Liquid chromatography mass spectrometry (LC-MS) was used to confirm the product’s identity. Mass spectra were collected using a H-Class Acquity UPLC system coupled to a Xevo

G2-S Q-ToF mass spectrometer (Waters Corp, Milford, MA), where a parent peak with m/z of

424.07662 was observed, which matches the theoretical m/z of xanthommatin [M +1] of 424.0775.

2.20 Computational methods

For each structure, a conformational search was performed for 10,000 conformers minimized with the OPLS3 force field, and an energy cut-off of 23.90 kcal mol–1 in Maestro130 included in the Schrodinger Suite of Programs. We selected the 10 lowest-energy conformers with a maximum atom deviation cutoff of 0.5 Å for optimization at the DFT level to identify the lowest energy conformer. All DFT calculations were performed using Gaussian 16.131 Stationary points were optimized with the B3LYP density functional, 6-311+G(d,p) basis set, and GD3BJ empirical dispersion correction.132 We included bulk solvation effects with the polarizable continuum model

(IEF-PCM) model (휺=78).133 Vibrational analysis was applied to each stationary point, indicating all transition state structures to have exactly one negative frequency, while all other stationary points had zero negative frequencies.

55 Chapter 3:

Contributions of Phenoxazone-Based Pigments to the Structure and Function of

Nanostructured Granules in Squid Chromatophores

3.1 Introduction

The ability to manipulate visible color in response to external stimuli such as light or applied voltage is observed in a diverse range of aquatic and terrestrial animals for communication, defense, maturation, or reproductive purposes.58, 64, 134-136 For example, integument pigments in the crab spider Misumena vatia facilitate the selective absorption of light producing white and yellow colors used for capturing prey or camouflaging from predators.58 On the other hand, highly ordered nanostructures localized within the translucent shell of the blue-rayed limpet Patella pellucida produce a bright blue color through the interference of incident light in a process that is used for visual communication.137 Cephalopods such as squid, cuttlefish, and octopus can display visible color across a broad spectral range using both highly ordered nanostructures and absorbing pigments,15, 23, 32, 138 making them some of the most sophisticated photonic systems in nature.

For instance, the squid Doryteuthis pealeii can manipulate its body coloration using two vertically arranged optical organs: the iridophore and the chromatophore.15-16, 20 Iridophores act as

Bragg stack reflectors that are composed of platelets containing reflectin, a structural protein assembled to produce constructive and destructive interference patterns in reflected light.32, 35, 139-

142 Chromatophores contain a tethered network of nanostructured pigmented granules that undergo a change in presented surface area to filter the light reflected from the underlying iridophore layer.20, 143-144 Several attempts to recapitulate the adaptive coloration of cephalopods through color-filled microfluidic channels,145 temperature-sensitive leucodye composites,146 voltage-

56 triggered fluorescent spiropyran mechanophores,147 and pressure-dependent electrochromic devices148 have illustrated the potential for such materials to impact flexible electronics, optical displays, or artificial skin. Despite the advancements by these bioinspired optoelectronic devices, existing strategies have yet to match the speed or dynamic range of visible coloration native to cephalopods. In most cases, the selected synthetic pigment acts as the limiting factor, as it regulates the optical feedback in response to external stimuli (temperature,146 pressure,148 or voltage147). In order to better inform systems designed to mimic adaptive coloration in cephalopods, we must first identify what is the composition of the chromatophore pigments, and how do they regulate light absorption and emission within the nanostructured granules in chromatophores.

Pigments in cephalopod chromatophores have previously been characterized as ommochromes,47, 149 a class of polycyclic aromatic metabolites of tryptophan that are comprised of substituted phenoxazone derivatives with signature UV-vis maxima centered at 360 and 480nm.49, 59, 150-151 However, the compositional analysis has not been reported.

Most of the information on ommochromes comes from other species. For instance, they are common pigment sources in the eyes, integumentary systems, organs, and eggs of arthropods.49, 58, 135, 152-153 Xanthommatin is the simplest and most common ommochrome and is formed (along with the reduced dihydro-xanthommatin, and ommatin subclasses) by the condensation of two hydroxykynurenine residues in the kynurenine pathway of tryptophan metabolism.49 It has been identified as a yellow pigment in dragonfly bodies, butterfly wings, and the hypodermis of spiders, exhibiting a red absorbance and bright blue- white fluorescence when exposed to UV light.64, 135, 154

The work we present here describes the extraction and the first mass spectrometric analysis of xanthommatin and decarboxylated xanthommatin from the nanostructured pigment granules in

57 chromatophores. We show that the removal of these pigments reduces the granular diameter and decreases visible color. When the extracted pigments are further purified and analyzed using mass spectrometry, we find that a unique combination of the carboxylated and decarboxylated forms of xanthommatin contribute to a broad range of colors exhibited by chromatophores, which span across the UV-vis range.

3.2 Results and Discussion

Adaptive coloration in squid D. pealeii (Figure 3.1a) is regulated by pigmented chromatophore cells which are layered on top of iridophore cells. While the structure-function properties of the iridophores have been well studied,15, 23, 32, 35, 138, 141 the chemical, nanoarchitectural, and optical properties of the chromatophore have not been fully detailed. To characterize these properties, squid chromatophores (Figure 3.1b) were first isolated, and their nanostructured pigment granules were removed and purified for analysis. Cephalopod pigments have previously been characterized as ommochromes,47, 149 which have a known solubility in polar organic solvents.58, 135, 154 To test whether ommochromes are present in the chromatophore granules, we adapted a protocol which was originally reported for the extraction of ommochromes from butterfly wings using acidic solutions of methanol (HCl-MeOH).135 The granules isolated from chromatophores were treated with HCl-MeOH, whereupon their original deep red color was extracted (Figure 3.1c). Scanning electron micrographs revealed that the HCl-MeOH extraction decreases the average diameter of the pigment granules from 583.5 ± 113.3 nm to 170.6 ± 34.6 nm

(Figure 3.1d). The ~70.8% reduction in diameter is accompanied by a loss in absorbance, indicating that the pigment has been successfully removed (Figure 3.1e). This is further supported with mass fraction analysis, where we observed a 57.6% (w/w) reduction in the mass of granules

58 post-extraction,60 suggesting the presence of pigment contributes to a significant portion of the granule.

Figure 3.1. Pigment extraction. (a) Squid D. Pealeii. (b) Bright field microscopic image of chromatophores. Scale bar is 1mm. (c) Pigment granules extracted from the chromatophores pre- and post- HCl-MeOH extraction. (d) Scanning electron microscopy analysis of pigment granules i) before HCl-MeOH extraction, scale bar is 300 nm and ii) after HCl-MeOH extraction, scale bar is 300 nm. (e) Bar graph showing the average pigment granule diameter pre- and post- acidic methanol extraction, n=75 granules, error is standard error of the mean. (f) Absorbance of suspended untreated pigment granules pre- and post- acidic methanol extraction. Bright field and electron microscopy images collected and processed by Dr. Sean Dinneen and Mr. Christopher DiBona.60 Reproduced from Ref. 60.

59 To determine whether the pigments isolated from chromatophore granules contain ommochromes, we first characterized their absorbance spectra using UV-vis spectrophotometry. Ommochromes are commonly identified by their characteristic absorption spectra.49 For instance, xanthommatin exhibits absorption bands in the UV region spanning from 225-240, 265-290, and 365-375 nm, with only one absorption maximum in the visible region centered around 435-485 nm.151 We asked whether the pigments extracted from chromatophore granules have similar UV-vis features. We observed a blue-shifted absorbance maximum in the soluble pigment supernatant (507 nm,

Figure 3.2a) when compared to the native granules (560 nm maximum, Figure 3.1f). The extracted pigment, presumably a mixture of compounds, was further separated via preparative normal phase thin layer chromatography (TLC), which revealed four optically distinct bands. The most mobile pigment (band i) had a retention factor of 0.55 ± 0.01, n

=22, and the least mobile pigment (band iv) had a retention factor of 0.09 ± 0.01, n =22,

(see figure 3.2a). After separation, the individual bands were extracted from the plate, and their absorbance spectra were measured (Figure 3.2b). Each band had common absorbance peaks of 294 and 365 nm. The least mobile band (iv) has an additional peak at 531 nm, and the most mobile bands (i and ii) have additional peaks at 493 nm (i) and 463 nm. Band iii has no distinguishable absorbance peaks outside of those described (Figure 3.2b). These data suggest that each isolated band contains a unique chromophore or set of chromophores that collectively contribute to the visible color observed in chromatophore granules.

60 Figure 3.2. Chromophore separation. (a) UV-vis data of pigment supernatant isolated from the HCl-MeOH extraction. The pigment supernatant is further separated using normal phase thin layer chromatography, resulting in four distinct bands, which are purified and analyzed. (b) UV-vis spectra of separated bands from the pigment extract, where red, blue, green, and purple colors represent pigment fractions from bands i, ii, iii, and iv, respectively.60 Images collected by Mr. Christopher DiBona. Reproduced from Ref. 60.

An additional optical feature of chromatophore pigment granules is their unique fluorescence emission. For instance, granules isolated from cuttlefish Sepia officinalis have a broad emission centered at 650 nm when excited at 410 nm.13 When the spherical structure of the granules is disrupted in the presence of concentrated sodium hydroxide, the far-red fluorescence emission blue-shifts and decreases significantly. This change has been correlated to a loss in the abundance of structural proteins such as reflectin and crystallin, suggesting that these proteins contribute to both granule structure and fluorescence emission.13 We asked whether the chromatophore pigments also contribute to the observed far-red fluorescence emission. To test this, we built a custom script to analyze the fluorescence profile of the granules before and after extraction as well as the separated pigment bands i-iv (Figure 3.3). As anticipated, the native pigment granules exhibited several emission peaks when excited at 390 nm, including local maxima at 490, 530, and

670 nm (Figure 3.3a). Upon pigment extraction, granules still maintained the emission

61 peaks, albeit with a decreased relative intensity (Figure 3.3b). The extracted pigments

(Figure 3.3c) and separated fractions (Figure 3.3di-iv) exhibited fluorescence spectra that differs from the granules. The far-red peaks were no longer observed when the soluble extracts were excited at 390 nm. Instead, the extracted pigment (Figure 3.3c) exhibits emission peaks centered at 600 and 630 nm only when excited at 530 nm. The separated pigment fractions also have no significant emission maxima, other than two poorly resolved peaks at ~430 and ~530 nm observed in band i (Figure 3.3di). The differences in excitation and emission maxima observed during pigment extraction and separation from the native chromatophore granules suggests that the source of far-red fluorescence is likely due to protein-based, structural components, supporting previous findings.13 The pigment alone does not give rise to the far-red fluorescence. It is only when coordinated within the original granule, where we observe this emission profile.

62 Figure 3.3. 3D fluorescence mapping. Spectra of granules (a) before and (b) after extraction, as well as (c) spectra of the extracted pigment supernatant. (d)TLC-separated fractions are shown for bands 1 (i), 2 (ii), 3 (iii), and 4 (iv). The granules, before and after pigment extraction, show multiple emission peaks when excited at about 386 nm. The supernatant and separated fractions do not. Reproduced from Ref. 60.

63 Given the differences in absorbance and fluorescence profiles of the extracted and separated pigments as compared to the native granules, we asked what is the composition of pigments contributing to the visible color of the cephalopod chromatophore granules. To test this, the separated pigment bands i-iv were collected, further purified using reverse phase HPLC, and analyzed using mass spectrometry. The monoisotopic masses of the ommatin and ommin subclasses hypothesized to be the main source of color in chromatophore granules were calculated and used to determine their presence and relative abundance within the chromatograms. In bands i-iv, we observed the abundance of both xanthommatin and decarboxylated (DC)-xanthommatin in different relative amounts.60

Their structural confirmation was based on high accuracy measurement of these molecules and their fragment ions from high-energy C-trap dissociation (HCD) (Figure 3.4a-b). As expected, single bond cleavage on the side-chain gives rise to all high abundance fragment ions. The 44 amu mass difference between similar fragment ions of xanthommatin and DC- xanthommatin further validates their structural assignment.

64 Figure 3.4. High-energy c-trap dissociation (HCD) spectra of molecules that match the mass of (a) decarboxylated xanthommatin and (b) xanthommatin. (c) Relative abundance of decarboxylated xanthommatin and xanthommatin from bands i, ii, iii, and iv, measured by their MS ion intensity. Data collected and processed by Mr. Christopher DiBona and Professor Feixia Chu.60 Reproduced from Ref. 60.

Previous studies show that some seasonal and maturation-related color changes in insects are made possible by the color dependent redox states of xanthommatin and DC- xanthommatin pigments, where a red color is associated with reduced forms, and yellow is

65 associated with oxidized forms.64 Considering the structural similarity between these two molecules, we measured their ion intensity and calculated their relative abundance in the reduced and oxidized states. In our analysis, we did not observe the reduced forms of either ommatin, possibly due to the ease with which these compounds oxidize in ambient conditions.49 Only the oxidized forms of each were detected, where an obvious trend emerges; the amount of DC- xanthommatin relative to xanthommatin decreases from band i to band iv (Figure 3.4c). When compared with the absorbance spectra collected from the separated pigment fractions in Figure 3.2c, the mass spec data reveal that the DC- xanthommatin enriched species exhibit a lighter visible color when compared to the darker xanthommatin enriched fractions. This information agrees with other reports which indicate possible correlations between the decarboxylated forms of xanthommatin and visible color outside of the known redox-dependent color changes.

In order to explain how the oxidation state of xanthommatin and decarboxylated xanthommatin may interact with the biological and optical aspects of the chromatophore, we first characterized the redox reaction. Based on stoichiometry, the reaction should proceed via proton-coupled electron transfer, with an overall addition or loss of 2 electrons and 2 protons. We used cyclic voltammetry to confirm that the redox reaction is reversible, to determine if the reaction is surface or diffusion limited, and to elucidate the dependence of the reaction on pH.

66

Figure 3.5. Electrochemical investigation of synthetic Xanthommatin (Xa). (a) Proposed chemical structures of Xa in both redox states. (b) Variations in redox behaviour associated with an increasing pH using a fixed scan rate of 100 mV/s. (i) pH of the Xa-electrolyte bath was varied, and CVs were collected on the third cycle; (ii) half-wave potentials of Xa (triangle, n = 3) plotted from (i) with an R2 = 0.998 ± 0.001. The potentials are compared to the control potassium hexacyanoferrate (circle), which was used to synthesize Xa. (c) The peak oxidative current of Xa was recorded at various scan rates in two different sodium phosphate solutions buffered at pH 1.7 and 7.4. The slopes taken from the linear fit of the log of the peak current versus the log of the scan rate at pH 1.7 (1.10 ± 0.1) and pH 7.4 (1.08 ± 0.1).155 Reprinted with permission from Ref. 159. Copyright 2018 American Chemical Society.

The shape of the CV shows that the reaction is, in fact, reversible and dependent on pH. Under neutral conditions (pH ≈ 7.4), the redox potential is -31 mV vs. Ag/AgCl, but under acidic conditions (pH≈ 1.7) the same reaction requires a potential of 310 mV. This

67 relationship, 62 mV/pH, is very close to the expected Nernstian behavior of 59 mV/pH, confirming a ratio of one proton to one electron in the reaction, according to equation 3.1.

퐻+ 퐸 = 퐸표 − 59 푚푉 ( ) ∗ 푝퐻 (3.1) 푛

These CVs also allow us to determine whether the reaction is diffusion limited or surface limited. As the scan rate for a CV is increased, the observed peak current should also increase. This is expected to be non-linear, with peak current (ip) increasing with the square root of the scan rate(ν), due to diffusion effects, as shown in the Randles-Sevcik equation (equation 3.2), where the number of electrons (n), the Faraday constant (F), the

0 bulk concentration (C ), temperature (T), gas constant (R), and diffusion coefficient (D0) remain constant.156

푛퐹휈퐷 1/2 푖 = 0.446푛퐹퐴퐶0 ( 0) (3.2) 푝 푅푇

Instead, we see a linear response with respect to scan rate, indicating that the xanthommatin adsorbs to the surface of the electrode.156 This is not significant to biological systems, but it does have implications in the production of electrochromic materials using xanthommatin.155

3.3 Conclusions

We report the first analytical confirmation of the presence of xanthommatin and decarboxylated xanthommatin in D. pealeii chromatophores. We adapted extraction protocols, which were originally optimized for insects and other arthropods, to selectively withdraw the pigments from the nanostructured chromatophore granules without destroying their spherical geometry. We use a combination of spectrophotometry, fluorescence spectroscopy, and mass spectrometry to show how unique combinations of xanthommatin and decarboxylated xanthommatin give rise to a broad range of colors that

68 span the UV-visible range. These data suggest a hierarchical mechanism for coloration in cephalopods originating from molecular components confined within the nanostructured granules of the chromatophore organs.

In the absence of the pigments, the spherical geometry of the granule is retained, as is its far-red fluorescence emission. In a previous report by Parker and coworkers, it was shown that when granular structure is destroyed in presence of concentrated sodium hydroxide, fluorescence emission decreases in a process that is correlated with the loss of structural proteins such as reflectin and crystallin.13 In the work described here, we find that when the pigment is extracted from the granule in acidic methanol, the spherical geometry is retained, albeit smaller in diameter and lighter in mass. This suggests that the pigment is not the sole contributor to the structure of the granule. Instead, a combination of structural proteins and small molecules help maintain the spherical structure and optical function of the granules. The ease with which the pigments can be removed from the granule without disrupting the spherical geometry suggests a core-shell interface, with the pigments in the core of the granule, and the proteins assembled at its shell.

Reflectin, for example, is a known structural protein which has the capabilities to assemble into platelets, thin films, fibers, or spheres depending on its reaction medium;29, 32, 106, 139-140, 142, 157 however, it has yet to be implicated as the coordinating matrix which assembles with pigment molecules to make up the supramolecular structures in the chromatophore. Future studies will be aimed at modeling the protein-pigment interactions to understand their role in color modulation and to inform the design of photonic devices that may better emulate the dynamic range of coloration exhibited by cephalopods.

Adapted with permission from Ref. 60. Copyright 2016 American Chemical Society.

69 Chapter 4: Dynamic Pigmentary and Structural Coloration Within Cephalopod Chromatophore Organs 4.1 Introduction

The richest and most diverse color patterns in the animal kingdom occur in organisms that have evolved elegant combinations of structural and pigmentary elements to manipulate light efficiently. For instance, octopus, squid, and cuttlefish have the ability to dynamically alter their appearance to quickly display a diverse range of camouflage and signaling10-11, 15, 22. This fast and dynamic adaptation involves the use of specialized dermal structures that modulate the animal’s appearance through multiple effects20. These structures include pigmentary chromatophore organs uppermost in the dermis as well as two classes of non-pigmentary (structural) coloration cell types: iridocytes that can specularly reflect nearly any color, appearing iridescent; and leucocytes that diffusely reflect all visible wavelengths at once, producing bright white. Iridocytes comprise protein platelets of a high-refractive-index protein reflectin that selectively reflect light via thin-film interference, producing a variety of iridescent colors spanning the visible spectrum23, 37,

42, 158. Leucocytes are also reflectin-based but mostly use microspheres to reflect diffuse white light43. Unlike the leucophores and iridophores, the composition of the light-interacting elements in the chromatophore is not yet well characterized.

Given their ability to work as dynamic color-filters within living tissue, the functional morphology of the chromatophore is of considerable interest from the viewpoints of both basic and applied science. Chromatophore neuromuscular organs comprise five cell types: nerves, glial cells, radial muscles, sheath cells, and the large central chromatocyte that is filled with a flexible cytoelastic sac containing nanostructured pigmented granules45. In Doryteuthis pealeii, each chromatocyte has 18-30 muscles radially arranged around its periphery that, upon neural stimulation, pull the pigmented cell outward into a flat colored disc16, 159. The mature chromatocyte

70 is occupied almost entirely with nanostructured granules containing ommochrome pigments and associated proteins that may help to maintain spatial cohesion among granules13, 47, 60. This combination of pigments and proteins maintains the color uniformity and richness of the chromatocytes65, 160 even as the cell expands into a very thin (2-4 granules thick) layer during actuation. Expansion of these systems is extremely fast (ca. 125 msec) due to the contractions of the radial muscle fibers attached to the chromatocyte. Thus, of particular interest is whether and to what extent light is being manipulated by both pigmentary and structural coloration within the cephalopod chromatophore to aid in its optical functions20, 23.

Although the composition of the pigments in the squid D. pealeii chromatophores has recently been verified as a combination of xanthommatin and decarboxylated xanthommatin60, the proteins within the chromatophore saccule, specifically those that might coordinate and couple with the pigments to aid in color filtering during actuation, remain unknown. Several proteins have been identified within or near the chromatophores including S-crystallin13, reflectin13, and r- opsin161-162. Crystallins are a diverse set of proteins found in the lens of animal eyes. One isoform,

S-crystallin, has been found in cephalopod eyes and skin52. This crystallin has also recently been shown to assemble into “patchy colloids” of varying density and refractive indices to prevent spherical aberration in the eyes of squid100. Another cephalopod lens crystallin found in the skin,

Ω-crystallin, is structurally homologous to aldehyde dehydrogenase, although it is enzymatically inactive51-52, 97. It is also the predominant isoform of crystallin found in the bioluminescent light organ of the Hawaiian bobtail squid Euprymna scolopes163. Although the S- and Ω- isoforms are compositionally dissimilar, they share (i) high refractive indices, (ii) optical transparency, and (iii) solubility in water, suggesting key functional roles in both the eyes and skin of the animals51-52, 96.

The structural protein reflectin is cephalopod-specific and aggregates into nanoparticles or self-

71 assembles into ribbons30, 34-35, contributing to its ability to scatter and reflect light that produces colorful iridescence or diffuse whiteness32, 164. Rhodopsin, a light-sensitive protein involved in phototransduction, has also been implicated in skin optical function, including the ability of the

(octopus) chromatophore system to respond to light even when physically isolated from cephalic input42, 161-162.

In this work we present evidence of an additional optical feature of cephalopod skin: intense colorful reflection from chromatophores. When combined with a systematic compositional and computational study, our data reveal that these organs are a remarkably refined system that produces dynamic coloration, stimulating reassessment of (i) how cephalopods are able to produce structural color in the absence of iridophore-like plates, and more generally (ii) how light might be manipulated to produce both pigmentary and structural coloration with richer photonic repertoires than previously recognized.

4.2 Results

4.2.1 Expanded Chromatophores Exhibit Intense Structural Color

We imaged chromatophores in living squid and in excised viable skin preparations using bright-field optical microscopy and observed colorful iridescent patches precisely located across the expanded surface of every color type of chromatophore (yellow, red or brown), especially with incident light at ca. 20-50° from the viewing angle (Figure 4.1). At the whole-animal level, we observed narrow streaks or patches of iridescence especially along the mantle and head of the squid (Figure 4.1A, Supplemental Video 1). All three colors of chromatophores displayed this iridescence, but particularly intense and colorful reflection was observed from the yellow chromatophores in live intact skin (Figures 1B-E). At high magnification, this phenomenon showed an iridescent, multiple-hue structural coloration quality even across a single

72 chromatophore. The colors progressed sequentially akin to Newton’s series (orders two and three, i.e. purple, blue, green, yellow, orange, red, Figure 4.1F)165. At times the reflectance appeared as a wrinkled membranous material (Figure 4.1E, Supplemental Video 3), reminiscent of shrink- wrapped plastic deforming under tension.

Figure 4.1 Iridescence produced in chromatophores of live squid. (A) Low power of mantle skin; arrows indicate narrow zones of iridescence that coincide with yellow chromatocytes (white arrows) and typical iridophores (green arrows). Scale bar is 3mm. (B, C) Arrows show yellow chromatophores under different lighting angles expressing different iridescence. Scale bars are 600 µm. (D) Yellow chromatophores showing iridescence that coincides with the exact expansion of the chromatocyte (arrow shows concentric variation in hue seen frequently). Scale bar is 300 µm. (E) Contrasting iridescence from a typical subjacent iridophore (green arrow) compared to a yellow chromatophore (white arrow). Scale bar is 1 mm. (F) A single yellow chromatophore showing granular and patchy iridescence. Scale bar is 100 µm. All microscopy images collected by Dr. Stephen Senft from the Marine Biological Laboratories.177 Reproduced from Ref. 177.

This reflectivity observed from the actuating chromatophores was initially mistaken for the iridescence long-known to be produced by underlying iridocytes (Figures 1A and E, green arrow).

73 However, upon closer examination we noted that the iridescence observed from the chromatophores was certainly different in location, size, shape, texture, and timing from the subjacent iridescing iridocytes. In D. pealeii, clusters of iridocytes sit below the uppermost chromatophore layer of the skin, where they could be readily recognized under the dissecting microscope from other cell types. Squid skin also contained large iridophore patches (several mm across, containing hundreds of iridocytes) that were located deeper in the dermis, widely spaced and at much lower densities than the chromatophores (Figures 1A, E, and Supplemental Video 1).

Moreover, iridophore iridescence exhibited minute facets, each reflecting a dominant wavelength

(based on numerous oriented platelets, operating singly and as Bragg stacks). Their hue did not vary with changing illumination angle like that observed from the iridescing yellow chromatophores (Figure 4.1). These data, along with additional observations regarding the effects of illumination and chromatophore expansion suggest the presence of structural coloration associated with the chromatophores that is completely separate from the iridophores.

4.2.2 Compositional Analysis of the Squid Chromatophore

We previously identified an abundance of both S- crystallins and various reflectin isoforms associated with semi-purified granules from cuttlefish chromatophores, suggesting that the granules were more than just chromogenic pigments13. However, the relationship between the pigments and proteins in chromatophores, including whether and how they coordinate and couple together to give rise to iridescence, remained unknown. We embarked on a detailed compositional analysis by first enzymatically isolating then manually sorting individual chromatophores by color from the dorsal mantle of five D. pealeii individuals, yielding ~700 yellow (n=2 animals), ~700 red (n=2 animals), and ~1000 brown chromatophore organs (n=3 animals; Figure 4.2A). The chromatophores were pooled by color, and proteins extracted from each set were proteolyzed and

74 analyzed using tandem mass spectrometry in conjunction with liquid chromatography (LC-

MS/MS, Figure 4.2B). Bioinformatic analysis of the LC-MS/MS data led to the identification of

469 putative protein entries translated from the transcriptome of squid D. pealeii chromatophores.

The isolated material included chromatocytes with their enclosed saccule containing the pigment granules, as well as surrounding membranes and, likely, sheath cells that may still be bound to some of the surrounding muscle fibers (Figure 4.2C). The function of identified protein entries was annotated through sequence homology using blast against the Uniprot non-redundant protein database. The identity of 412 (all but 57) of these proteins matched positively to known proteins, constituting the annotated proteome specific to squid chromatophores (Table A.1.). We compared the relative amounts of each identified protein in different color chromatophore cells using a semi- quantitative spectrum counting approach166-167 and grouped them by biological functions (Figure

4.2B). We assessed the relative abundance of proteins from different color chromatophore cells by a protein abundance index, where peptide counts were scaled by the number of predicted peptides produced by tryptic digestion168-169, as detailed in the methods section. Total peptide count was normalized between chromatophore types to avoid sample-loading bias. Due to the difficulty of collecting sufficient material for replicate experiments, our assessment of relative protein abundances is less accurate for the low abundance proteins170.

75

Figure 4.2 Mass spectrometric analysis of chromatophore proteins. (A) Enzymatically isolated squid dorsal mantle chromatophores, retaining the plasma membrane of the variously colored chromatocytes and their surrounding sheath cells (sc). Scale bar is 6.5 µm. (B) Proteins identified and categorized by function in the yellow, red, and brown pigment cells in squid D. pealeii dorsal and ventral mantle. (C) Typical morphological arrangement of chromatophores in squid skin; note the radial muscles visible around the central retracted brown chromatophore. Scale bar is 500 µm. (D) Categorized proteins identified within the squid chromatophore granules, pigment extracted granules (e.g. the granule “shell”), and the extracted pigment along with representative SEMs (scale bar = 500 nm) and optical image of extracted pigment. Bright field images collected by Dr. Stephen Senft from MBL; electron micrographs collected by Dr. Amrita Kumar.177 Reproduced from Ref. 177.

The most abundant individual protein was Ω- crystallin, whose normalized peptide count was 290, 170, and 256 for the yellow, red, and brown cells, respectively, while S-crystallin was the second most abundant protein in red and brown cells, with normalized peptide counts of 101 and 144. Compared to the total number of proteins, the various crystallin isoforms made up only

10-13% of all peptides detected, while the various metabolism-related proteins, loosely characterized as any protein involved in the production or catabolism of biomolecules, collectively

76 represented 30-40% of the detected peptides (Figure 4.2B, Table A.1.). We observed color-linked variations in nearly every category of protein and two major trends emerged from our analysis: the brightest chromatophores, the yellows, had the most reflectins (isoforms A1, A2, B1, C1, and 3) which have been known to contribute to enhanced reflectivity of surfaces37, 43; while the darkest chromatophores, the browns, had more crystallin (Ω- plus S-), the latter of which has been suggested to enhance absorption of light via scattering in other systems99. The protein content in the red cells represented a combination of the browns and yellows. It is noteworthy that only four proteins were identified as glutathione S-transferases (GSTs) (Table A.1.). While S-crystallins and

GSTs are homologous to one another with similar primary structures52, BLASTP comparisons indicated that they can be clearly differentiated from one another. In our categorization, we labeled

S-crystallins as such when the identified polypeptide sequence had >90% coverage and >60% identity similar to that of S-crystallin (see Table A.1.). When taken together, the presence of these various protein classes indicated that the sacculus and the surrounding cell membrane network were composed of a heterogeneous population of proteins responsible for signaling, structural, metabolic, and optical functions that contribute to the complexity of the chromatophore organ.

In order to ensure that these results were statistically significant, additional analysis was performed using the QSpec statistical framework developed by Choi, Fermin, and Nesvizhskii.170

This framework was developed to assist in the analysis of label-free mass spectrometry based

“shotgun proteomics,” where a mixture of proteins are digested, then identified using LC/MSMS, using a hierarchical Bayes method to identify differentially expressed proteins.170-171 Because

QSpec compares samples pairwise, chromatophore types were placed into pairs: brown and red, brown and yellow, and red and yellow. Peptide counts for each pair were processed by the QSpec software, which also normalized each set by protein content and scaled individual proteins by

77 molecular weight. For each protein in each pair of chromatophore types, this provides both the fold change, a measure of the change in protein abundance, as well as the calculated false discovery rate (FDR). For each detected protein, the -log10(FDR) was plotted against the log2(Fold Change) and colored by assigned category (Figure 4.3). For the majority of reflectin and crystallin isoforms detected, the FDR is less than 5%, indicating statistical significance, even though the absolute change in protein abundance is not always large.

Figure 4.3. QSpec analysis showing both scale and significance of differences in protein expression, as measured by spectral count MS/MS data. The three chromatophores are compared pairwise (Brown vs. Red, Brown vs. Yellow, and Red vs. Yellow) to show how protein abundance differs between brown, red, and yellow chromatophores. For each protein detected in more than one chromatophore, significance is expressed as the negative log of the false discovery rate (- log10(FDR), while the magnitude of the differential expression between the two types of chromatophore is shown as the log base two of the fold change (log2((Fold Change)). Reproduced from Ref. 177.

78 4.2.3 Crystallin But Not Reflectin Is found Amidst Pigment Granules

The presence of reflectins and crystallins in the isolated chromatophores has significant ramifications. It confirms a previous finding of both proteins in S. officinalis brown chromatophores13 and indicates that the percentage of reflectin varies among the different color types of chromatophores. However, it was still unclear whether the proteins were localized within or outside the chromatocytes or their saccules. Thus, we attempted to target a more selective sub- section of the chromatocyte: the isolated and purified pigment granules. Here, we did not separate the granules based on chromatophore color; instead, we collected whole skin sections across the dorsal and ventral regions of four animals, similar to previous reports60. Our extraction protocol also enabled us to selectively extract the pigment from the granules, leaving behind a granule

“shell” that was colorless but still appeared spherical under scanning electron microscopy (Figure

4.2D).

We compared the compositions of the intact granules (Table S2), pigment-extracted granules (the granule “shell,” Table S3), and the extracted pigment (Table S4), where we posited that the remaining shell was composed of proteins that remained largely unaffected by the acid hydrolysis used to extract the pigment. We assessed the relative abundance of identified proteins within each sample using spectral counting, similar to how we treated the chromatophore LC-

MS/MS data (Figure 4.2D). While peptide count was scaled by the expected number of fragments produced by tryptic digestion, overall protein loading between samples was not normalized. Based on protein identity, they were either classified by function, as shown in Figure 4.2D, or as a contaminant including skin-surface bacterial ribosomal proteins or enzymes used in sample preparation (Tables A.2., A.3., and A.4.). After accounting for contaminating proteins, we identified 65, 11, and 8 individual proteins in the granules, pigment-extracted granules, and the

79 pigment-only samples, respectively. The intact granules comprised a variety of proteins associated with metabolism, the cell membrane, and the extracellular matrix, while the shell portion of the granules and the extracted pigment contained a subset of the granule-associated proteins and were both comparatively enriched with Ω -crystallin. S-crystallin and reflectin were not found in any of these three sample types.

4.2.4 Reflectin Molecules Are Distributed Throughout Sheath Cells

Our proteomic data indicated that the granule-only samples included Ω- crystallin but not reflectin. This observation was perplexing considering that the whole-cell data in Figure 4.2B and

Table A.1. showed multiple reflectin isoforms present in the manually selected chromatophore material. Thus, we asked whether we could identify where reflectin(s) might reside within the overall chromatophore organ. We obtained polyclonal antibodies that, as confirmed by Western blots, targeted the reflectin A1 and A2 isoforms (other isoforms are known to exist, but specific antibodies against them are not yet available). We used immunocytochemistry and confocal microscopy to identify the cellular distribution of these epitopes in squid skin and observed the strongest signal of reflectin within the sheath cells, which completely envelop the chromatocyte in all planes (Figures 5A-C), including particularly dense label seen as a mass on the flat surface of the chromatophore. Reflectin was found consistently localized to the sheath cells in chromatophores from both mantle and fin areas of the squid across replicate trials, tissue samples, and chromatophore color. Occasionally, but not routinely, fluorescent signal was observed close to the sacculus and radial muscle fibers.

80

Figure 4.4. Anatomical localization of reflectin to sheath cells. (A) Reflectin (confocal, secondary antibody in green, arrows) was present surrounding the edge of the chromatocyte and the apical portions of the radial muscles (M). Scale bar is 50 µm; (B) Reflectin (confocal, dual secondary antibodies in lavender; 405nm and 568nm fluorophores were applied simultaneously to reduce the potential ambiguity associated with auto-fluorescence of the tissue) often extended into the spaces between adjacent apical radial muscles. Scale bar is 25 µm; (C) Reflectin distribution was frequently punctate, and in addition to coating the muscles and the edge of the chromatocyte, reflectin was also present over its surface. Note that the chromatocyte section was very thin (in comparison with the confocal depth of field of the 0.45NA 20x objective used here), but the label consistently showed on its outside, rather than within its sacculus. Scale bar is 25 µm; (D) Confocal section of a chromatophore imaged by autofluorescence with the sheath cells highlighted in yellow for better visualization (scale bar is 50 µm). Sheath cells completely enveloped the pigment sac in all dimensions, as illustrated in the electron micrograph in (E); scale bar is 12 µm. All images collected by Dr. Stephen Senft from the MBL. Reproduced from Ref. 177.

Sheath cells are an integral component of chromatophore organs, yet their function has not been studied. Historically they were presumed to provide a flexible buffer zone separating the pigment saccule from the adjacent connective tissue, providing low resistance against actuation45,

172. While no definitive individual morphology has yet been assigned to these cells, our own light

81 and electron micrographs confirmed that sheath cells are highly folded membranous cells that wrap around the entire chromatocyte with multiple layers, including the proximal portion of the radial muscles near the pigment sacculus potentially also playing a nutritive role, since no mitochondria or other metabolic machinery was seen in our electron micrographs of chromatocytes (Figures

4.4D and E). While the total number of cells that ensheath a chromatocyte remains unknown, stains with 4′,6-diamidino-2-phenylindole (DAPI) revealed many nuclei adjacent to the positively stained reflectin regions, which were also adjacent to the chromatocyte and in between radial muscles (Figures 4.4A-C). Some of our confocal and EM images indicate that these cells may be flattened and highly infolded sheets with variable electron densities and textures, suggesting a structural role that could contribute to the regional variation of the chromatophore iridescence

(Figure 4.4E).

One of the most interesting aspects of this system is that structural coloration is observed without clear underlying ultrastructural electron-dense reflectin-filled lamellae similar to the signature plate-like stacks of cephalopod iridocytes36-37. However, thin film interference in general is known to generate iridescence173. Thus, to better understand the potential source(s) of the reflected coloration localized to the chromatophores, we used a multilayer interference equation24 to model the dependence of visible color on the nanolayered elements (e.g., sheath cell membranes and cytoplasm) surrounding the chromatophore, where we hypothesized that these structures may contribute to the perceived iridescence. To numerically evaluate this possibility, we measured and averaged the height of 292 cytoplasm layers (da as 116 ± 102 nm) and 237 sheath cell membrane layers (distances, db, as 71 ± 14 nm). We calculated a range of reflected color associated with the variable cytoplasm layer heights (da), which were up to ~3x larger than the sheath cell membrane layers. Lower wavelengths were predicted for the smaller spacings (approaching ~100 nm),

82 suggesting that as the chromatophore expands during actuation, the cytoplasm layer would likely spread out, decreasing its layer height to lead to a blue-shifted reflected color. As the chromatophore relaxes (e.g., retracts in diameter), the cytoplasm layers thicken back up, effectively increasing the layer thicknesses towards higher wavelengths. Together, these estimations provide one potential mechanism describing the dynamic range of visible color presented in the chromatophores during actuation. Dynamic morphology of other components in the chromatophore where refractive index contrast may be present (such as interfaces between sheath cell and chromatocyte or between the sacculus and contained granules) in principle could generate similar optical effects.

4.3 Discussion

The optical properties of cephalopod skin continue to surprise. Chromatophore organs in squid skin, thought to be exclusively absorptive and pigmentary, are also structurally reflective at select viewing angles. This previously unexplored feature is accompanied by the discovery of reflectin surrounding the chromatocyte and crystallin residing within the chromatocyte. Reflectin has long been recognized as an essential component that generates static or neurally tunable iridescence (iridocytes) as well as exceptionally bright broadband white (leucocytes) in skin layers below the chromatophores42-43. On the other hand, the S- isoform of crystallin in squid eyes is present as a colloidal gel with a refractive index gradient that enables the squid lens to counteract spherical aberration100, while the Ω- isoform of crystallin is present in the photophore of the bobtail

E. scolopes, believed to relieve oxidative stress in its light organ163. Using an annotated proteome specific to squid chromatophores, we confirm the presence of these proteins in the dermal chromatophores, spatially segregated within the pigment sac (Ω- crystallin) and surrounding it

(reflectin).

83 It is unusual for the elements of pigmentary and structural coloration to interact as intimately as seen in the squid chromatophore. In select species of butterflies, spiders, reptiles and birds173-174, these elements can be observed within the same tissue but they are generally located in discrete cell layers or assemblages (or, if blended within one tissue as with butterfly scales173, they produce a single visual effect). For instance, various skin colors are produced in day geckos via iridophore cells interspersed with melanophores and erythrocytes; however, this combination generates static color patterns only175. In contrast, the dynamic coloration of the panther chameleon is made possible by two distinct layers of cells, one that features iridophores that modulate brightness and a second layer that uses either iridophores or pigmented cells to control hue176. The spatial segregation of color sources into cell layers described in these systems is not dissimilar from squid skin viewed at the large scale, where there is a topmost layer of chromatophores and a subjacent layer of iridophores. However, it is now clear that the squid chromatophore organ, unlike most of these other systems, has closely intertwined pigmentary and structural coloration components at the molecular and cellular level to produce dynamic colors that can appear both richly pigmentary or brightly iridescent.

The abundance of Ω-crystallin (a structural homolog of aldehyde dehydrogenase) and its affinity to xanthommatin suggest dual roles for this protein that contribute to (i) the nanostructure of the granule and (ii) the stabilization and/or sequestration of xanthommatin in the granule, which would otherwise be a toxic byproduct of tryptophan metabolism. Both features are likely contributors to pigment color retention, light absorption, and scattering, as the chromatophore experiences a change in presented surface area during actuation. From a subsequent computational analysis,177 we posit a mechanism describing the coacervation of the protein-pigment complex into granules—a process that has eluded researchers for decades. Based on the distinct electrostatic

84 gradients on the protein surface, we reason that the positively charged regions of one tetramer may stabilize and bind to the negatively charged regions of an adjacent protein (monomer or tetramer) leading to structural associations that may propagate to form the nanostructured granules in the chromatophore. While further investigations are required to support this position, our findings reveal a unique combination of Ω-crystallin and pigments in the granules that may play an important role in regulating color within the chromatocyte.

Outside the chromatocyte, we describe a mode of reflection via structural color originating in part from the chromatophore sheath cells, which are shown here to contain reflectin. Since the chromatocyte and sheath cell membranes are deformable yet closely apposed, chromatophore actuation that expands the pigment cell into a flattened disc might automatically produce a parallel membrane geometry conducive to wavelength-selective interference. While this hypothesis is speculative, it is supported by our direct optical observation of chromatophore iridescence in the expanded state with certain narrow angles of lighting, and by the observation of reflectin aggregations intimately associated within sheath cells tightly surrounding the chromatocyte. It is particularly noteworthy that no platelets (as in iridocytes) or spheres (as in leucosomes) have been found within the sheath cells or elsewhere in or near the chromatophore organ in our numerous micrographs. Instead, at high optical magnification the reflectin antigenicity in fixed tissue is observed as small granular vesicles dispersed in seemingly disordered fashion amidst the convoluted sheath cells, which themselves must morph radically during the 15x areal expansion of the pigmented chromatocyte when it is maximally distended by the radial muscles for full color dispersal. Considering that reflectins were recently reported to contain no transmembrane domains178, we speculate that one or more reflectin isoforms could be condensed (dynamically) along or within the numerous folded sheath cell membrane invaginations that appose each other

85 with sub-micron gaps outside the chromatocyte. In principle, such folds might constitute a geometrical compartmentalization for reflectin that when flattened could yield structural color using incident photons and those optically filtered by the absorptive granules in the pigment saccule and reflecting upwards. This is consistent with a previous study in which self-assembled films of reflectin exhibited structural color due to thin-film interference, which is highly dependent on film thickness35.

How might reflectin influence light from the chromatophore? Since the sheath cells completely surround the pigment granules in the chromatocyte, light that enters the expanded chromatophore from the surface will pass through the sheath cells (and hence encounter the reflectins) to interact with pigment granules before they exit vertically to produce reflection via scattering, effectively doubling its effect. In other cases, light could enter chromatophores from below (the skin is often transparent) to also pass through sheath cells on both sides of the pigment sac. In such cases this structural coloration could, for example, amplify the brightness of the pigments, as we have seen in biomimetic analogues of chromatophores160 and have illustrated in

Figure 4.5.

86

Figure 4.5 Predicted optical effects within and around the chromatophore organs. The associated optical phenomena proposed based on our current and previous (see reference 17) findings are represented as (A) back scatter, (B) refraction, (C) forward scatter, (D) absorption (of non-yellow wavelengths), (E) multi-layer interference, and (F) diffuse scattering, where (G) represents the radial muscle fibers; (H) represents the sheath cells; (I) represents the cytoplasm of the sheath cells; (J) represents individual granules; (K) represents the a collection of granules within the yellow chromatocyte; and (L) represents an iridophore, deeper in the dermis. For simplicity, only yellow colored chromatophores are illustrated here. Figure created by Dr. Sean Dinneen. Reproduced from Ref. 177.

While our understanding of the reflectin family is still growing (by the last count there are dozens of isoforms), we observed only 5 in our MS/MS analysis (A1, A2, B1, C1, and 3) and 2

(A1 and A2) from our immunocytochemical labeling. Because the antibodies used in our study were highly unlikely to have revealed all reflectins, a near-future task will require in situ labeling of chromatophores using RNA-based probes to better understand the spatial distribution of these proteins.

87 When taken together, the compositional, computational, and optical analyses of the squid chromatophore presented here – from molecules to the whole organ - provide insights into the ultrastructure and chemical composition of these organs. While cephalopod chromatophores have been thought to be solely pigmentary organs for decades20, 43, 45, 179, our findings demonstrate an additional photonic mechanism via structural iridescence that could conceivably be used to enhance the dynamic range of color presented by the squid, or to provide an additional specular visual cue contextually detectable by conspecifics, predators or prey. Together, these findings along with the knowledge that approximately 15 additional isoforms of reflectins exist in cephalopods with functions yet to be explored, suggest there is still much more to be learned about these complex systems, including a better understanding of molecular mechanisms regulating these dynamic biophotonic processes especially as it pertains to the future designs of engineered materials with dynamic optical capabilities.

Adapted with permission from Ref. 177. Text and indicated figures are licensed under a Creative

Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

88 Chapter 5: A Scalable Method to Synthesize the Xanthommatin

Biochrome Via an Electro Catalyzed Oxidation of Tryptophan Metabolites

5.1 Introduction

Xanthommatin, a natural phenoxazinone biochrome, is abundant in insects, arachnids, and cephalopods.49, 58-60 It was first identified in insect eyes, where it was hypothesized to act as a screening pigment to protect the more sensitive structures of the eye from photobleaching.49 It has also been discovered in certain dragonflies, where upon sexual maturity, males change in color from yellow to red due to the reduction of xanthommatin.64 More recently, xanthommatin has been confirmed as a pigment in the chromatophores of some cephalopods, where its high extinction coefficient, high refractive index, and redox-dependent color change contribute to the broad range of rich colors found in these dermal organs.60, 65 These animals synthesize xanthommatin via the kynurenine metabolic pathway, an adaptation that doubles as a method to sequester otherwise toxic tryptophan.49, 180 First, tryptophan is enzymatically converted into 3-hydroxykynurenine (3-OHK), and then two equivalents of 3-OHK dimerize to form xanthommatin, which can be isolated in pigmented granules or within membrane-bound organelles.80-81, 87, 181 Attempts have been made in the laboratory to recapitulate xanthommatin biosynthesis using potassium ferricyanide

(K3[Fe(CN)6]) as an oxidizing agent to facilitate the six electron oxidation to dimerize 3-OHK and produce dihydroxanthommatin, which is converted to xanthommatin in aerobic environments in a two electron oxidation.49, 62, 76 The most widely used purification of xanthommatin from ferricyanide is solid phase extraction (SPE), which can successfully remove most but not all of the iron from the product, making quality control of xanthommatin difficult to manage.

89 This difficulty has inspired the development of alternative synthetic methods focused on simplicity in design and in purification. In this report, we investigate how replacing ferricyanide with a fixed oxidative potential impacts the formation, stability, and purity of xanthommatin from

3-OHK. Distinguished from previous reports,182 we introduce a strategy to easily scale the system using reticulated vitreous carbon (RVC) working electrodes and show how this process leads to a product that can be more readily purified on a preparatory scale. Because of the low cost of the material as well as its high surface area to volume ratio, RVC electrodes have been shown to improve reaction times compared to two-dimensional electrodes.183-184 This improved synthesis may increase the appeal of xanthommatin for future materials applications, as well as allow some additional insight into the reaction mechanism and kinetics.

In addition to the scalable synthetic approach presented here, we also propose a mechanism guided by previous literature on the oxidative dimerization of ortho-aminophenols (OAPs),185 describing xanthommatin formation from 3-OHK. While it has been proposed that xanthommatin synthesis involves an electrooxidative cascade, similar to OAP dimerization,63, 186 these studies do not explicitly describe the intramolecular cyclization necessary for xanthommatin synthesis. We report the first mechanism supported by both experimental data as well as computational methods for this step of the reaction. We use density functional theory (DFT) to model possible reactions involving both closed- and open-shell mechanistic pathways. This analysis lays an important framework for understanding this one-pot reaction, opening a pathway towards high throughput synthesis of xanthommatin and other asymmetric OAPs.

90 5.2 Results

It is known that xanthommatin is formed by the oxidative dimerization of 3-OHK, during which 8 molar equivalents of electrons and protons, as well as one molar equivalent of ammonia, are lost (Figure 5.1A).55 We ask whether the electrooxidation of 3-OHK could similarly produce xanthommatin, albeit in a one-pot reaction under pH neutral, aqueous conditions (Figure 5.1B).

As we apply a fixed oxidative potential, the measured current informs us of the reaction progress.(Figure 5.1C).187 Since the current is proportional to the concentration of oxidizable species at the surface of the electrode, the decrease of current over time suggests that the starting material, 3-OHK, is being consumed more quickly than it is replenished by stirring. As the reaction reaches 90 minutes of applied potential, the current drops below 5% of the maximum and appears to approach a steady state, indicating that the reaction is approximately complete. When integrated as a function of time,184 the current also provides the total charge (Q) involved in the reaction, allowing us to calculate the number of electrons (n) lost per molecule of xanthommatin synthesized, using the Faraday constant (F) and the mass of 3-OHK used (ma, 0.010 g), as shown in equation 5.1:

푄 2∗224.21 푛 = ∗ ( ) (5.1) 퐹 푚푎

In this reaction, the final charge is calculated to be equivalent to 6.8 electrons per molecule of xanthommatin, which is less than the 8 electrons predicted by the stoichiometric conversion of 3-

OHK to xanthommatin. However, the value of 6.8 electrons is expected for the six electron oxidation that produced dihydroxanthommatin, assuming some faradaic inefficiency.

91

Figure 5.1. Summary of xanthommatin electrosynthesis (A) Overall reaction scheme. xanthommatin is formed by the dimerization of 3-OHK, requiring the oxidative loss of eight electrons and protons, as well as the loss of ammonia. (B) An illustration of the reaction cell. The reaction cell consists of an RVC working electrode (WE), a silver-silver chloride (Ag/AgCl) reference electrode (RE), and a platinum counter electrode (CE) separated from the bulk solution by glass frit. (C) Controlled-potential chronocoulometry showing reaction progress. The flow of current between the WE and CE is recorded and integrated as a function of time to determine the total charge passed through the system. To evaluate product conversion over time, aliquots are removed and characterized, as described in the methods section (Figure 5.2). As 3-OHK is converted into xanthommatin, the intensity of its absorption maxima (λmax) increases and shifts from 370 nm to 450 nm, suggesting an increase in conjugation over time (Figure 5.2Ai). The λmax at 450 nm increases from 0.04 to

1.40 A.U. with no effective change after 90 minutes (Figure 5.2Aii). CV is also used to determine the relative amount of 3-OHK. 3-OHK exhibits an irreversible oxidation peak at 0.3 V vs. Ag/AgCl

(Figure 5.2Bi), which decreases over time, again with very little change after 90 minutes (Figure

5.2Bii); whereas, xanthommatin shows a reversible oxidation with anodic and cathodic peaks at

10 mV and -76 mV vs. Ag/AgCl, respectively. The oxidized product was purified by semipreparative HPLC and confirmed to be xanthommatin by both NMR (Figure 5.3) and LC-

MS. Together, these measurements confirm that the applied oxidative potential used here is

92 sufficient to convert 3-OHK to xanthommatin, and that the reaction does not continue past 90 minutes.

Figure 5.2. Spectroscopic and voltammetric characterization of electrosynthesis aliquots. (Ai) UV/Vis spectral measurements before and after 90 minutes of applied potential show peak absorbances consistent with 3-OHK, at 370 nm, and xanthommatin, at 454 nm. (Aii) Absorbance at 454 nm increases over time as xanthommatin is produced and approaches a maximum as the reaction completes, corresponding to the intense color change of the bulk solution (inset). (Bi) CV reveals a change in the redox properties after electrosynthesis. (Bii) Following peak oxidation current at approximately 0.3V shows the consumption of 3-OHK over time.

93

Figure 5.3. Nuclear magnetic resonance (NMR) spectrum of xanthommatin in deuterated dimethyl sulfoxide (D6-DMSO) with trifluoroacetic acid (TFA). (A) Wide view showing major solvent peaks (TFA, D6-DMSO). (B) Enhanced view focused on xanthommatin specific peaks. Identified xanthommatin peaks are indicated by number, corresponding to the numbered proton on the xanthommatin structure. Contaminating solvent peaks are labeled.

94 The synthesis is easily scalable (Figure 5.4), where we demonstrate the conversion of 30 mg of 3-OHK in a 4.5 mM solution under an applied potential. We observe that the current initially remains steady, around 6.4 mA, for the first hour. After 60 minutes, the current decreases exponentially, until it reaches a steady state of 0.7 mA, approximately 10% of the maximum current. The final charge, 47.1 C, shows that the equivalent charge of 7.1 electrons per molecule of xanthommatin are used in the reaction, according to equation 1, which is consistent with the smaller reaction size. After SPE, the yield is 92%, representing a facile method to scale the synthesis of xanthommatin while minimizing the number of reagents used to do so.

Figure 5.4. Controlled-potential chronocoulometry of the scaled-up reaction. 30 mg of xanthommatin were dissolved in 30 mL of 0.1M phosphate buffer at pH 7.2. The current (black) is measured as a fixed potential of 0.5 V is applied to the stirred solution, and the charge (red), calculated as the integral of the current as a function time. The reaction approaches completion after 215 minutes of applied potential.

95 Given the ease of scaling the reaction, we next interrogated the mechanisms potentiating xanthommatin formation under the applied electrochemical field. Previous work suggests that

OAPs dimerize through an alternating series of oxidation reactions and nucleophilic addition reactions.185 This process has been observed with OAPs, which dimerize to form aminophenoxazinones,80, 181, 188-189 as well as with catechols and para-aminophenols, where a reactive quinone or quinone imine is produced by oxidation and a second species acts as a nucleophile to attack it.190-195 3-OHK likely follows the same mechanisms to achieve the phenoxazinone structure, before a final cyclization reaction that results in xanthommatin (Figure

5.5). In this mechanism, 3-OHK is oxidized, forming a benzoquinone imine (BQMI), and nucleophilically attacked by the amine of a second, unoxidized 3-OHK in a Michael-like addition, followed by a facile proton transfer to regenerate the aminophenol moiety. This process is effectively repeated with a second oxidation and Michael addition of the phenol oxygen to the unsaturated carbon β to the quinone oxygen. A third oxidation produces the amino-phenoxazinone core of xanthommatin.

96

Figure 5.5. A proposed mechanism, supported by literature,63, 181, 185, 188, 196 describing the initial dimerization of 3-OHK to form the phenoxazinone core of xanthommatin via alternating oxidation and coupling reactions. These reactions are analogous to previously described reactions occurring in the synthesis of amino-phenoxazone from ortho-aminophenols.

97 Since this initial cyclization reaction is analogous to the well-studied dimerization of OAP, we decided to explore the mechanism of the second cyclization step to afford xanthommatin from the phenoxazinone intermediate. We used DFT calculations to differentiate the competing cyclization mechanisms (Michael-like vs. SN2). Figure 5.6 (A and B) shows the possible cyclization mechanisms The phenoxazinone intermediate (6a) has previously been detected experimentally via LC-MS coupled with an electrochemical detection cell.182 Two likely routes to dihydroxanthommatin 6a include an intramolecular SN2 reaction or a Michael-like addition. The

SN2 reaction involves the phenoxazinone amine nucleophilically attacking the α-carbon of the amino acid chain, displacing the primary amine (Figure 5.6A). The Michael-like addition involves the amine on the amino acid chain to attack the carbon adjacent to the phenoxazinone amine (β to the imine), followed by the elimination of ammonia (Figure 5.6B). Additionally, these reactions are occurring under oxidative conditions, and stable xanthommatin and H2xanthommatin radicals have been previously detected, suggesting that the open-shell variations of these reactions, a radical substitution and a radical addition, must also be considered.182, 186, 197

98

Figure 5.6. Computational exploration of two mechanisms of xanthommatin formation from the phenoxazinone intermediate. (A) Proposed SN2 mechanism. (B) Proposed Michael-like addition. (C) Potential energy surface for the SN2 and radical substitution mechanisms. D) Potential energy surface for the Michael-like addition. The free energy values are computed using H2O B3LYP/6-311+G(d,p) IEF-PCM . Both the closed-shell SN2 reaction (solid lines) and the open-shell radical substitution reaction (dashed lines). In order to determine the preferred mechanism of the second cyclization step towards xanthommatin, we computed the energies of the transition states, intermediates, and products of these. As such, we first computed the phenoxazinone intermediates 6a and 6b, which correspond to the likely protonation states of the intermediate in the reaction buffer. The zwitterionic form,

6a, is expected to be the major species at pH 7.4 and is used as the starting material for the substitution reactions. While the deprotonated amino acid amine should be in a minor species in

99 the reaction buffer at pH 7.4, the observed cyclization suggests that this reactive species should be capable of promoting the Michael-like cyclization. The activation free energies for the closed- and

-1 open-shell substitution mechanisms are 53.5 and 68.6 kcal mol , respectively. These barriers are prohibitively high because of the poor leaving group character of amines; this pathway can be eliminated as a viable mechanistic path. We turned our attention to the Michael-like addition to determine if this mechanism was more accessible. Figure 5.7 shows the structure of intermediate

6b, transition structures and activation free energies for the closed- and open-shell pathways.

Figure 5.7. Calculated geometry of the transition states for the closed shell Michael addition (TS (6b→8) - S0) and open shell Michael-like addition (TS (6b→8) - D1). The N-C bonds formed in the transition states are indicated with a dashed line, and the bond length is shown. Additionally, the relative Gibbs free energy of the activated complexes are shown below each structure. The optimized geometries and free energy values are computed using B3LYP/6-311+G(d,p) IEF- PCMH2O. The activation free energies of TS(6b→8)-S0 and TS(6b→8)-D1 are 20.4 and 4.7 kcal

-1 mol respectively. TS(6b→8)-S0 and TS(6b→8)-D1 feature similar bond lengths of 1.97 Å

(Figure 5.4). The 14.7 kcal mol–1 preference for the open-shell pathway arises from the increased reactivity associated with the oxidized 6b. The immediate product (8-S0) of the Michael-like addition reaction is not energetically favored, with a free energy of 18.0 kcal mol-1 relative to the starting material (6b- S0), but the formation of the final product, H2Xa- S0, is strongly exergonic,

-1 with an overall ΔGrxn of -30.7 kcal mol , driving the forward. The open-shell reaction also appears

100 -1 to be reversible, as the free energy of the immediate product (8-D1) is 3.7 kcal mol relative to the starting material (6b-D1), but, again, the proceeding loss of ammonia and rearrangement to

-1 produce H2Xa- D1 are irreversible, with a final free energy of –55.6 kcal mol .

While this new information regarding the mechanism may not be useful for further optimizing the synthesis of xanthommatin, it does suggest a synthetic pathway towards new phenoxazinones. xanthommatin can be used as an electrochromic pigment, but it only features a single color change, from red to yellow. Having knowledge of the cyclization mechanism allows for the intelligent design of new phenoxazinones by modifying parts of the alkyl chain of 3-OHK in ways that preserve the cyclization reaction, but still alter the solubility or electrochromic properties of the final product. By modifying, for example, the methylene bridge in the alkyl chain of 3-OHK with an electron withdrawing group, the cyclization reaction should still occur, but the redox potential and optical properties of the final product will be altered.

5.3 Conclusions

Starting with the known oxidation properties of 3-OHK, we show an improved synthetic method to produce xanthommatin. By replacing six molar equivalents potassium ferricyanide, with an applied potential, we reduce the complexity of the synthesis while reducing its economic and environmental impacts. We show that the reaction is quick and continues to completion, using both spectrophotometric and electrochemical techniques. DFT calculations were used to determine which of the several possible mechanisms could give rise to the xanthommatin pyrido group, resulting in the conclusion that a Michael addition or radical addition are both likely to occur at reasonable rates, depending on whether the starting material is a radical. Together, these findings describe the application of electrosynthesis to produce xanthommatin and provide a theoretical framework for the development of new asymmetric phenoxazinones.

101 Chapter 6: Conclusions and Recommendations

Understanding the structure-function relationships of pigment-based nanostructures can provide insight into the molecular mechanisms behind biological signaling, camouflage, or communication experienced in cephalopods. This insight then allows for the development of new bioinspired optical materials that recapitulate the properties of cephalopod dermis – specifically the ability to dynamically alter coloration in a quick and accurate manner on a flexible, biocompatible substrate. In order to better understand the molecular mechanisms behind cephalopod chromatophore function, we first determined the pigmentary contributions to coloration. Then we explored the composition of the structural elements to infer how they might contribute to coloration as well. Finally, we performed a new method of xanthommatin synthesis as a first step towards the large-scale development of bioinspired materials.

In squid D. pealeii, combinations of phenoxazone-based pigments are identified as the source of visible color within the nanostructured granules that populate dermal chromatophore organs. In the absence of the pigments, granules experience a reduction in diameter with the loss of visible color, suggesting important structural and functional features. These results implicate a hierarchical mechanism for the bulk coloration in cephalopods originating from the molecular components confined within in the nanostructured granules of chromatophore organs.

In order to determine the identity of the non-pigmentary components, we performed a proteomic study of D. pealeii chromatophores and pigment granules. We find the lens protein Ω- crystallin interfacing tightly with pigment molecules within the granules and within the extracted pigment. We also report the discovery of structural coloration emanating in precise register with expanded pigmented chromatocytes. Concurrently, using an annotated squid chromatophore proteome together with microscopy collected from collaborators at the Marine Biological

102 Laboratories in Woods Hole, MA, we identify a likely biochemical component of this reflective coloration as reflectin proteins distributed in sheath cells that envelop each chromatocyte. These findings offer fresh perspectives on the intricate biophotonic interplay between pigmentary and structural coloration elements tightly co-located within the same dynamic flexible organ - a feature that may help inspire the development of new classes of engineered materials that change color and pattern.

While chromaotophore emulating devices have been recently produced, both by Alon

Gorodetsky and John Rossiter,104, 107 our findings represent additional mechanisms present within the chromatophores which may prove useful for the production of new bioinspired materials. The use of xanthommatin, as in the native granules, imparts additional functionality, such as electrochromism and a high refractive index.65, 155 The effect of pigment encapsulation within a nanoparticle may improve absorbance though additional scattering effects. Incorporation of tunable strucutral coloration through the use of thin-film intereference adds additional dimensions of color adaptability. These additional insights into the biological mechanisms invoved in chromatophore coloration may lead to improved optical properties and functions of artificial chromatophores and new optical materials otherwise inspired by chromatophores.

As an initial step towards the fabrication of these new materials, we looked to improve the synthesis of xanthommatin. Xanthommatin is a naturally occurring broad-spectrum dye present not only in cephalopods but also in a wide variety of arthropods. In these animals, xanthommatin is enzymatically produced via the oxidative cyclization of 3-hydroxykynurenine (3-OHK), a metabolite of tryptophan, but it can also be synthesized in vitro using an iron-based oxidizing agent. While the optoelectronic properties (e.g., high refractive index, redox-sensitive color) of both natural and synthetic forms of xanthommatin have inspired its use in materials, its synthesis

103 faces challenges, both in terms of scalability and ease of purification. We describe a procedure to synthesize xanthommatin via electrochemical oxidation of 3-OHK, which offers both economic and ecological advantages over the traditional method. Supported by a mechanistic assessment using density functional theory calculations, we describe a feasible pathway for the electro- catalyzed oxidation and cyclization of xanthommatin based on the loss of eight electrons and protons from two molar equivalents of 3-hydroxykynurenine within a one-pot reaction. Our results provide insight into a method of xanthommatin synthesis that could conceivably be scaled up for future materials applications and/or adapted to the production of other phenoxazinones synthesized from ortho-aminophenols.

104 References 1. Whitesides, G. M., Bioinspiration: something for everyone. Interface Focus 2015, 5 (4), 20150031. 2. McConnell, I.; Li, G.; Brudvig, G. W., Energy Conversion in Natural and Artificial Photosynthesis. Chem. Biol 2010, 17 (5), 434-447. 3. Khan, A. A.; Tahir, M., Recent advancements in engineering approach towards design of photo-reactors for selective photocatalytic CO2 reduction to renewable fuels. J. CO2 Util. 2019, 29, 205-239. 4. Yang, W.; Lopez, P. J.; Rosengarten, G., Diatoms: Self assembled silica nanostructures, and templates for bio/chemical sensors and biomimetic membranes. Analyst 2011, 136 (1), 42- 53. 5. Knecht, M. R.; Wright, D. W., Functional analysis of the biomimetic silica precipitating activity of the R5 peptide from Cylindrotheca fusiformis. Chem. Commun. 2003, 24, 3038-3039. 6. Pamirsky, I. E.; Golokhvast, K. S., Silaffins of diatoms: from applied biotechnology to biomedicine. Mar. Drugs 2013, 11 (9), 3155-3167. 7. Knecht, M. R.; Wright, D. W., Amine-Terminated Dendrimers as Biomimetic Templates for Silica Nanosphere Formation. Langmuir 2004, 20 (11), 4728-4732. 8. Gomes, C. M.; Deravi, L. F., Self-assembling extracellular matrix proteins as materials for the condensation of silica nanostructures. RSC Advances 2016, 6 (97), 95337-95341. 9. Hanlon, R. T.; Messenger, J. B., Cephalopod behaviour. Cambridge University Press: 2018. 10. Buresch, K. C.; Mäthger, L. M.; Allen, J. J.; Bennice, C.; Smith, N.; Schram, J.; Chiao, C.-C.; Chubb, C.; Hanlon, R. T., The use of background matching vs. masquerade for camouflage in cuttlefish Sepia officinalis. Vision Res. 2011, 51 (23), 2362-2368. 11. Mäthger, L. M.; Chiao, C.-C.; Barbosa, A.; Hanlon, R. T., Color matching on natural substrates in cuttlefish, Sepia officinalis. J. Comp. Physiol. A 2008, 194 (6), 577. 12. Aristotle, I., Historia Animalium, english translation by D'Arcy Wenthworth Thompson. Oxford: Clarendon Press: 1910. 13. Deravi, L. F.; Magyar, A. P.; Sheehy, S. P.; Bell, G. R. R.; Mäthger, L. M.; Senft, S. L.; Wardill, T. J.; Lane, W. S.; Kuzirian, A. M.; Hanlon, R. T.; Hu, E. L.; Parker, K. K., The structure-function relationships of a natural nanoscale photonic device in cuttlefish chromatophores. J. R. Soc. Interface 2014, 11, 20130942. 14. Kreit, E.; Mäthger, L. M.; Hanlon, R. T.; Dennis, P. B.; Naik, R. R.; Forsythe, E.; Heikenfeld, J., Biological versus electronic adaptive coloration: how can one inform the other? J. Roy. Soc. Interface 2013, 10 (78), 20120601. 15. Mäthger, L. M.; Denton, E. J.; Marshall, N. J.; Hanlon, R. T., Mechanisms and behavioural functions of structural coloration in cephalopods. J. Roy. Soc. Interface 2009, 6, S149-S163. 16. Messenger, J. B., Cephalopod chromatophores: neurobiology and natural history. Biol. Rev. 2001, 76, 473-528. 17. Langridge, K. V., Cuttlefish use startle displays, but not against large predators. Anim. Behav. 2009, 77 (4), 847-856. 18. Norman, M. D.; Finn, J.; Tregenza, T., Dynamic mimicry in an Indo-Malayan octopus. Proc. Biol. Sci. 2001, 268 (1478), 1755-1758. 19. Adamo, S. A.; Hanlon, R. T., Do cuttlefish (Cephalopoda) signal their intentions to conspecifics during agonistic encounters? Anim. Behav. 1996, 52 (1), 73-81.

105 20. Cloney, R. A.; Brocco, S. L., Chromatophore Organs, Reflector Cells, Iridocytes and Leucophores in Cephalopods. Am. Zool. 1983, 23, 581-592. 21. Land, M. F., The physics and biology of animal reflectors. Prog. Biophys. Mol. Biol. 1972, 24, 75-106. 22. Mäthger, L. M.; Shashar, N.; Hanlon, R. T., Do cephalopods communicate using polarized light reflections from their skin? J. Exp. Biol. 2009, 212 (14), 2133-2140. 23. Mäthger, L. M.; Hanlon, R. T., Malleable skin coloration in cephalopods: selective reflectance, transmission and absorbance of light by chromatophores and iridophores. Cell Tissue Res. 2007, 329, 179-186. 24. Kinoshita, S.; Yoshioka, S.; Miyazaki, J., Physics of structural colors. Rep. Prog. Phys. 2008, 71 (7), 076401. 25. Kinoshita, S.; Yoshioka, S., Structural Colors in Nature: The Role of Regularity and Irregularity in the Structure. ChemPhysChem 2005, 6 (8), 1442-1459. 26. Hiroshi, N.; Noriko, O.; Ryozo, F., Light-reflecting properties of the iridophores of the neon tetra, Paracheirodon innesi. Comp. Biochem. Physiol., Part A Mol. Integr. Physiol. 1990, 95 (3), 337-341. 27. Gur, D.; Leshem, B.; Pierantoni, M.; Farstey, V.; Oron, D.; Weiner, S.; Addadi, L., Structural Basis for the Brilliant Colors of the Sapphirinid Copepods. J. Am. Chem. Soc. 2015, 137 (26), 8408-8411. 28. DeMartini, D. G.; Krogstad, D. V.; Morse, D. E., Membrane invaginations facilitate reversible water flux driving tunable iridescence in a dynamic biophotonic system. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (7), 2552-2556. 29. Tao, A. R.; DeMartini, D. G.; Izumi, M.; Sweeney, A. M.; Holt, A. L.; Morse, D. E., The role of protein assembly in dynamically tunable bio-optical tissues. Biomaterials 2010, 31 (5), 793-801. 30. Guan, Z.; Cai, T.; Liu, Z.; Dou, Y.; Hu, X.; Zhang, P.; Sun, X.; Li, H.; Kuang, Y.; Zhai, Q.; Ruan, H.; Li, X.; Li, Z.; Zhu, Q.; Mai, J.; Wang, Q.; Lai, L.; Ji, J.; Liu, H.; Xia, B.; Jiang, T.; Luo, S.-J.; Wang, H.-W.; Xie, C., Origin of the Reflectin Gene and Hierarchical Assembly of Its Protein. Curr. Biol. 2017, 27 (18), 2833-2842.e6. 31. Williams, L. W., The anatomy of the common squid Loligo pealii, Lesueur. Brill: 1909. 32. Crookes, W. J.; Ding, L. L.; Huang, Q. L.; Kimbell, J. R.; Horwitz, J.; McFall-Ngai, M. J., Reflectins: The unusual proteins of squid reflective tissues. Science 2004, 303 (5655), 235- 238. 33. Ordinario, D. D.; Phan, L.; Walkup Iv, W. G.; Jocson, J.-M.; Karshalev, E.; Hüsken, N.; Gorodetsky, A. A., Bulk protonic conductivity in a cephalopod structural protein. Nat. Chem. 2014, 6 (7), 596-602. 34. Naughton, K. L.; Phan, L.; Leung, E. M.; Kautz, R.; Lin, Q.; Van Dyke, Y.; Marmiroli, B.; Sartori, B.; Arvai, A.; Li, S.; Pique, M. E.; Naeim, M.; Kerr, J. P.; Aquino, M. J.; Roberts, V. A.; Getzoff, E. D.; Zhu, C.; Bernstorff, S.; Gorodetsky, A. A., Self-Assembly of the Cephalopod Protein Reflectin. Adv. Mater. 2016, 28 (38), 8405-8412. 35. Kramer, R. M.; Crookes-Goodson, W. J.; Naik, R. R., The self-organizing properties of squid reflectin protein. Nat. Mater. 2007, 6 (7), 533-538. 36. Ghoshal, A.; DeMartini, D. G.; Eck, E.; Morse, D. E., Experimental determination of refractive index of condensed reflectin in squid iridocytes. J. Roy. Soc. Interface 2014, 11 (95), 20140106.

106 37. Ghoshal, A.; DeMartini, D. G.; Eck, E.; Morse, D. E., Optical parameters of the tunable Bragg reflectors in squid. J. Roy. Soc. Interface 2013, 10 (85). 38. Roger, T. H.; Lydia, M. M.; George, R. R. B.; Alan, M. K.; Stephen, L. S., White reflection from cuttlefish skin leucophores. Bioinspiration Biomim. 2018, 13 (3), 035002. 39. Hanlon, R. T.; Cooper, K. M.; Budelmann, B. U.; Pappas, T. C., Physiological color change in squid iridophores. Cell Tissue Res. 1990, 259 (1), 3-14. 40. Hanlon, R. T., The functional organization of chromatophores and iridescent cells in the body patterning of Loligo plei (Cephalopoda: Myopsida). Malacologia 1982, 23 (1), 89-119. 41. Cooper, K. M.; Hanlon, R. T.; Budelmann, B. U., Physiological color change in squid iridophores. Cell Tissue Res. 1990, 259 (1), 15-24. 42. Izumi, M.; Sweeney, A. M.; DeMartini, D.; Weaver, J. C.; Powers, M. L.; Tao, A.; Silvas, T. V.; Kramer, R. M.; Crookes-Goodson, W. J.; Mäthger, L. M.; Naik, R. R.; Hanlon, R. T.; Morse, D. E., Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid. J. R. Soc. Interface 2010, 7(44), 549-560. 43. Mäthger, L. M.; Senft, S. L.; Gao, M.; Karaveli, S.; Bell, G. R. R.; Zia, R.; Kuzirian, A. M.; Dennis, P. B.; Crookes-Goodson, W. J.; Naik, R. R.; Kattawar, G. W.; Hanlon, R. T., Bright White Scattering from Protein Spheres in Color Changing, Flexible Cuttlefish Skin. Adv. Funct. Mater. 2013, 23 (32), 3980-3989. 44. DeMartini, D. G.; Ghoshal, A.; Pandolfi, E.; Weaver, A. T.; Baum, M.; Morse, D. E., Dynamic biophotonics: female squid exhibit sexually dimorphic tunable leucophores and iridocytes. J. Exp. Biol. 2013, 216 (19), 3733-3741. 45. Cloney, R. A.; Florey, E., Ultrastructure of cephalopod chromatophore organs. Z. Zellforsch. Mikrosk. Anat. 1968, 89 (2), 250-280. 46. Reiter, S.; Hülsdunk, P.; Woo, T.; Lauterbach, M. A.; Eberle, J. S.; Akay, L. A.; Longo, A.; Meier-Credo, J.; Kretschmer, F.; Langer, J. D.; Kaschube, M.; Laurent, G., Elucidating the control and development of skin patterning in cuttlefish. Nature 2018, 562 (7727), 361-366. 47. Van den Branden, C.; Decleir, W., A study of the chromatophore pigments in the skin of the cephalopod Sepia officinalis. Biologische Jaarb 1976, 44, 345-352. 48. Froesch, D.; Packard, A., Octopus chromatophores accumulate nickel. Experientia 1979, 35 (6), 828-830. 49. Linzen, B., The Tryptophan → Ommochrome Pathway in Insects. In Advances in Insect Physiology, J.E. Treherne, M. J. B. a. V. B. W., Ed. Academic Press: 1974; Vol. 10, pp 117-246. 50. Schwinck, I., Vergleich des Redox-Pigmentes aus Chromatophoren und Retina von Sepia officinalis mit Insektenpigmenten der Ommochromgruppe. Verh. Deut. Zool. Ges.(Zool. Anz. 19, Suppl. 71-75) 1956. 51. Chiou, S. H., A novel crystallin from octopus lens. FEBS Letters 1988, 241 (1-2), 261- 264. 52. Tomarev, S. I.; Piatigorsky, J., Lens Crystallins of Invertebrates. Eur. J. Biochem. 1996, 235 (3), 449-465. 53. Liddell, H. G.; Scott, R., A Greek-English Lexicon. Jones, S. H. S., Ed. Clarendon Press: Oxford, 1940. 54. Becker, E., Die Pigmente der Ommin- und Ommatingruppe, eine neue Klasse von Naturfarbstoffen. Naturwissenschaften 1941, 29 (16), 237-238. 55. Butenandt, A., Ommochrome. III. The synthesis of xanthommatin. Liebigs Ann. 1954, 588, 106-116.

107 56. Butenandt, A., Schiedt, U., Biekert, E., & Kornmann, P., Über Ommochrome, I. Mitteilung. Isolierung von Xanthommatin, Rhodommatin und Ommatin C aus den Schlupfsekreten von Vanessa urticae. Eur. J. Org. Chem. 1954, 586 (1), 217-228. 57. Needham, A. E., The significance of zoochromes. Springer Science & Business Media: 2012; Vol. 3. 58. Riou, M.; Christidès, J.-P., Cryptic Color Change in a Crab Spider (Misumena vatia): Identification and Quantification of Precursors and Ommochrome Pigments by HPLC. J. Chem. Ecol. 2010, 36 (4), 412-423. 59. Insausti, T. C.; Casas, J., The functional morphology of color changing in a spider: development of ommochrome pigment granules. J. Exp. Biol. 2008, 211, 780-789. 60. Williams, T. L.; DiBona, C. W.; Dinneen, S. R.; Jones Labadie, S. F.; Chu, F.; Deravi, L. F., Contributions of Phenoxazone-Based Pigments to the Structure and Function of Nanostructured Granules in Squid Chromatophores. Langmuir 2016, 32, 3754-3759. 61. Tomoda, A.; Yoneyama, Y.; Yamaguchi, T.; Shirao, E.; Kawasaki, K., Mechanism of Coloration of Human Lenses Induced by Near-Ultraviolet-Photo-Oxidized 3-Hydroxykynurenine (With 1 color plate). Ophthalmic Res. 1990, 22 (3), 152-159. 62. Vazquez, S.; Garner, B.; Sheil, M. M.; Truscott, R. J. W., Characterisation of the major autoxidation products of 3-hydroxykynurenine under physiological conditions. Free Radic. Res. 2000, 32 (1), 11-23. 63. Bolognese, A.; Liberatore, R.; Riente, G.; Scherillo, G., Oxidation of 3- hydroxykynurenine. A reexamination. J. Heterocycl. Chem. 1988, 25 (4), 1247-1250. 64. Futahashi, R.; Kurita, R.; Mano, H.; Fukatsu, T., Redox alters yellow dragonflies into red. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (31), 12626-12631. 65. Dinneen, S. R.; Osgood, R. M.; Greenslade, M. E.; Deravi, L. F., Color Richness in Cephalopod Chromatophores Originating from High Refractive Index Biomolecules. J. Phys. Chem. Lett. 2017, 8 (1), 313-317. 66. Romero, Y.; Martínez, A., Antiradical capacity of ommochromes. J. Mol. Mod. 2015, 21 (8), 220. 67. Varadkar, S.; Surtees, R., Glutaric aciduria type I and kynurenine pathway metabolites: A modified hypothesis. J. Inherit. Metab. Dis. 2004, 27 (6), 835-842. 68. Sawada, H.; Nakagoshi, M.; Mase, K.; Yamamoto, T., Occurrence of ommochrome- containing pigment granules in the central nervous system of the silkworm, Bombyx mori. Comp. Biochem. Physiol. B 2000, 125 (3), 421-428. 69. Douglas, A. E., Phloem-sap feeding by animals: problems and solutions. J. Exp. Bot. 2006, 57 (4), 747-754. 70. Lovelace, M. D.; Varney, B.; Sundaram, G.; Lennon, M. J.; Lim, C. K.; Jacobs, K.; Guillemin, G. J.; Brew, B. J., Recent evidence for an expanded role of the kynurenine pathway of tryptophan metabolism in neurological diseases. Neuropharmacology 2017, 112, 373-388. 71. Reed, R. D.; Nagy, L. M., Evolutionary redeployment of a biosynthetic module: expression of eye pigment genes vermilion, cinnabar, and white in butterfly wing development. Evol. Dev. 2005, 7 (4), 301-311. 72. Zhang, H.; Lin, Y.; Shen, G.; Tan, X.; Lei, C.; Long, W.; Liu, H.; Zhang, Y.; Xu, Y.; Wu, J.; Gu, J.; Xia, Q.; Zhao, P., Pigmentary analysis of eggs of the silkworm Bombyx mori. J. Insect Physiol. 2017, 101, 142-150. 73. Lewis-Ballester, A.; Forouhar, F.; Kim, S.-M.; Lew, S.; Wang, Y.; Karkashon, S.; Seetharaman, J.; Batabyal, D.; Chiang, B.-Y.; Hussain, M.; Correia, M. A.; Yeh, S.-R.; Tong, L.,

108 Molecular basis for catalysis and substrate-mediated cellular stabilization of human tryptophan 2,3-dioxygenase. Sci. Rep. 2016, 6, 35169. 74. Han, Q.; Robinson, H.; Li, J., Biochemical identification and crystal structure of kynurenine formamidase from Drosophila melanogaster. Biochem. J. 2012, 446 (2), 253-260. 75. Varma, S. D.; Hegde, K. R., Kynurenine-induced photo oxidative damage to lens in vitro: protective effect of caffeine. Mol. Cell. Biochem. 2010, 340 (1), 49-54. 76. Figon, F.; Casas, J., Ommochromes in invertebrates: biochemistry and cell biology. Biol. Rev. 2019, 94, 156-183. 77. Théry, M.; Casas, J., The multiple disguises of spiders: web colour and decorations, body colour and movement. Philos. Trans. Royal Soc. B 2009, 364 (1516), 471-480. 78. Liu, S. H.; Luo, J.; Yang, B. J.; Wang, A. Y.; Tang, J., Karmoisin and cardinal ortholog genes participate in the ommochrome synthesis of Nilaparvata lugens (Hemiptera: Delphacidae). Insect Sci. 2017, 26, 35-43. 79. Osanai-Futahashi, M.; Tatematsu, K. i.; Futahashi, R.; Narukawa, J.; Takasu, Y.; Kayukawa, T.; Shinoda, T.; Ishige, T.; Yajima, S.; Tamura, T.; Yamamoto, K.; Sezutsu, H., Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis. Heredity 2015, 116, 135. 80. Le Roes-Hill, M.; Goodwin, C.; Burton, S., Phenoxazinone synthase: what's in a name? Trends Biotech. 2009, 27 (4), 248-258. 81. Li, J.; Beerntsen, B. T.; James, A. A., Oxidation of 3-hydroxykynurenine to produce xanthommatin for eye pigmentation: a major branch pathway of tryptophan catabolism during pupal development in the Yellow Fever Mosquito, Aedes aegypti. Insect Biochem. Mol. Biol. 1999, 29 (4), 329-338. 82. Vogliardi, S.; Bertazzo, A.; Comai, S.; Costa, C. V. L.; Allegri, G.; Seraglia, R.; Traldi, P., An investigation on the role of 3‐hydroxykynurenine in pigment formation by matrix‐assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2004, 18 (13), 1413-1420. 83. The UniProt Consortium, UniProt: the universal protein knowledgebase. Nucleic Acids Res. 2017, 45 (D1), D158-D169. 84. Letunic, I.; Bork, P., Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 2016, 44 (W1), W242-W245. 85. Phillips, J. P.; Forrest, H. S.; Kulkarni, A. D., Terminal Synthesis of Xanthommatin in Drosophila melanogaster. III. Mutational Pleiotropy and Pigment Granule Assocoation of Phenoxazinone Synthetase. Genetics 1973, 73 (1), 45-56. 86. Shoup, J. R., The Development of Pigment Granules in the Eyes of Wild Type and Mutant Drosophila melanogaster. J. Cell Biol. 1966, 29 (2), 223-249. 87. Insausti, T. C.; Casas, J., Turnover of pigment granules: Cyclic catabolism and anabolism of ommochromes within epidermal cells. Tissue Cell 2009, 41 (6), 421-429. 88. Rasgon, J. L.; Scott, T. W., Crimson: A Novel Sex-Linked Eye Color Mutant of Culex Pipiens L. (Diptera: Culicidae). J. Med. Entomol. 2004, 41 (3), 385-391. 89. Abraham, E. G.; Sezutsu, H.; Kanda, T.; Sugasaki, T.; Shimada, T.; Tamura, T., Identification and characterisation of a silkworm ABC transporter gene homologous to Drosophila white. Mol. Gen. Genet. 2000, 264 (1), 11-19. 90. Khan, S. A.; Reichelt, M.; Heckel, D. G., Functional analysis of the ABCs of eye color in Helicoverpa armigera with CRISPR/Cas9-induced mutations. Sci. Rep. 2017, 7, 40025.

109 91. Higgins, C. F.; Linton, K. J., The ATP switch model for ABC transporters. Nat. Struct. Mol. Biol. 2004, 11, 918-926. 92. Martel, R. R.; Law, J. H., Purification and properties of an ommochrome-binding protein from the hemolymph of the tobacco hornworm, Manduca sexta. J. Biol. Chem. 1991, 266 (32), 21392-21398. 93. Zinovieva, R. D.; Piatigorsky, J.; Tomarev, S. I., O-Crystallin, arginine kinase and ferritin from the octopus lens. Biochim. Biophys. Acta 1999, 1431 (2), 512-517. 94. Zigler, J. S.; Sinha, D., βA3/A1-crystallin: More than a lens protein. Prog. Retin. Eye Res. 2015, 44, 62-85. 95. Horwitz, J., Alpha-crystallin can function as a molecular chaperone. Proc. Natl. Acad. Sci. U.S.A. 1992, 89 (21), 10449-10453. 96. Tan, W.-H.; Cheng, S.-C.; Liu, Y.-T.; Wu, C.-G.; Lin, M.-H.; Chen, C.-C.; Lin, C.-H.; Chou, C.-Y., Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein. Sci. Rep. 2016, 6, 31176. 97. Zinovieva, R. D.; Tomarev, S. I.; Piatigorsky, J., Aldehyde dehydrogenase-derived omega-crystallins of squid and octopus. Specialization for lens expression. J. Biol. Chem. 1993, 268 (15), 11449-55. 98. Chuang, C.-C.; Wu, S.-H.; Chiou, S.-H.; Chang, G.-G., Homology Modeling of Cephalopod Lens S-Crystallin: A Natural Mutant of Sigma-Class Glutathione Transferase with Diminished Endogenous Activity. Biophys. J. 1999, 76 (2), 679-690. 99. Chang, H.-C.; Lin, T.-L.; Chang, G.-G., Molecular Basis for the Polymerization of Octopus Lens S-Crystallin. Biophys. J. 2000, 78 (4), 2070-2080. 100. Cai, J.; Townsend, J. P.; Dodson, T. C.; Heiney, P. A.; Sweeney, A. M., Eye patches: Protein assembly of index-gradient squid lenses. Science 2017, 357 (6351), 564-569. 101. Horwitz, J.; Ding, L.; Vasiliou, V.; Cantore, M.; Piatigorsky, J., Scallop lens Ω-crystallin (ALDH1A9): A novel tetrameric aldehyde dehydrogenase. Biochem. Biophys. Res. Commun. 2006, 348 (4), 1302-1309. 102. Piatigorsky, J.; Kozmik, Z.; Horwitz, J.; Ding, L.; Carosa, E.; Robison, W. G.; Steinbach, P. J.; Tamm, E. R., Ω-Crystallin of the Scallop Lens: A Dimeric Aldehyde Dehydrogenase Class 1/2 Enzyme-Crystallin. J. Biol. Chem. 2000, 275 (52), 41064-41073. 103. Sone, Y.; Ekdunge, P.; Simonsson, D., Proton Conductivity of Nafion 117 as Measured by a Four‐Electrode AC Impedance Method. J. Electrochem. Soc. 1996, 143 (4), 1254-1259. 104. Xu, C.; Stiubianu, G. T.; Gorodetsky, A. A., Adaptive infrared-reflecting systems inspired by cephalopods. Science 2018, 359 (6383), 1495-1500. 105. Phan, L.; Ordinario, D. D.; Karshalev, E.; Walkup Iv, W. G.; Shenk, M. A.; Gorodetsky, A. A., Infrared invisibility stickers inspired by cephalopods. J. Mater. Chem. C 2015, 3 (25), 6493-6498. 106. Phan, L.; Walkup IV, W. G.; Ordinario, D. D.; Karshalev, E.; Jocson, J.-M.; Burke, A. M.; Gorodetsky, A. A., Reconfigurable Infrared Camouflage Coatings from a Cephalopod Protein. Adv. Mater. 2013, 25 (39), 5621-5625. 107. Rossiter, J.; Conn, A.; Cerruto, A.; Winters, A.; Roke, C., Colour gamuts in polychromatic dielectric elastomer artificial chromatophores. Proc. SPIE. 2014, 9056, 905620. 108. Pikul, J. H.; Li, S.; Bai, H.; Hanlon, R. T.; Cohen, I.; Shepherd, R. F., Stretchable surfaces with programmable 3D texture morphing for synthetic camouflaging skins. Science 2017, 358 (6360), 210-214.

110 109. Yan, J.; Wu, S.-T.; Cheng, K.-L.; Shiu, J.-W., A full-color reflective display using polymer-stabilized blue phase liquid crystal. Appl. Phys. Lett. 2013, 102 (8), 081102. 110. Rossiter, J.; Yap, B.; Conn, A., Biomimetic chromatophores for camouflage and soft active surfaces. Bioinspiration Biomim. 2012, 7 (3), 036009. 111. Pelrine, R.; Kornbluh, R.; Kofod, G., High-Strain Actuator Materials Based on Dielectric Elastomers. Adv. Mater. 2000, 12 (16), 1223-1225. 112. Fishman, A.; Catsis, S.; Homer, M.; Rossiter, J. M., Smart squid skin: patterns in networks of artificial chromatophores. Proc. SPIE 2018. 10594, 105941S. 113. Allen, J. J.; Bell, G. R. R.; Kuzirian, A. M.; Hanlon, R. T., Cuttlefish skin papilla morphology suggests a muscular hydrostatic function for rapid changeability. Journal of Morphology 2013, 274 (6), 645-656. 114. Sherma, J.; Fried, B., Handbook of thin-layer chromatography. CRC press: 2003; Vol. 89. 115. Goldstein, J., Practical scanning electron microscopy: electron and ion microprobe analysis. Springer Science & Business Media: 2012. 116. Skoog, D. A.; Holler, F. J.; Crouch, S. R., Principles of instrumental analysis. Cengage learning: 2017. 117. Chu, F.; Shan, S. O.; Moustakas, D. T.; Alber, F.; Egea, P. F.; Stroud, R. M.; Walter, P.; Burlingame, A. L., Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16454-9. 118. Zeng-Elmore, X.; Gao, X.-Z.; Pellarin, R.; Schneidman-Duhovny, D.; Zhang, X.-J.; Kozacka, K. A.; Tang, Y.; Sali, A.; Chalkley, R. J.; Cote, R. H.; Chu, F., Molecular Architecture of Photoreceptor Phosphodiesterase Elucidated by Chemical Cross-Linking and Integrative Modeling. J. Mol. Biol. 2014, 426 (22), 3713-3728. 119. Weber, K.; Osborn, M., The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 1969, 244 (16), 4406-4412. 120. Alon, S.; Garrett, S. C.; Levanon, E. Y.; Olson, S.; Graveley, B. R.; Rosenthal, J. J. C.; Eisenberg, E., The majority of transcripts in the squid nervous system are extensively recoded by A-to-I RNA editing. eLife 2015, 4, e05198. 121. MacManes, M. D., Establishing evidenced-based best practice for the de novo assembly and evaluation of transcriptomes from non-model organisms. bioRxiv 2016, 035642. 122. MacManes, M. D.; Eisen, M. B., Improving transcriptome assembly through error correction of high-throughput sequence reads. PeerJ 2013, 1, e113. 123. MacManes, M. D., On the optimal trimming of high-throughput mRNA sequence data. Front. Genet. 2014, 5, 13. 124. Simão, F. A.; Waterhouse, R. M.; Ioannidis, P.; Kriventseva, E. V.; Zdobnov, E. M., BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 2015, 31 (19), 3210-3212. 125. Smith-Unna, R.; Boursnell, C.; Patro, R.; Hibberd, J. M.; Kelly, S., TransRate: reference- free quality assessment of de novo transcriptome assemblies. Genome Res. 2016, 26 (8), 1134- 1144. 126. Guan, S.; Burlingame, A. L., Data processing algorithms for analysis of high resolution MSMS spectra of peptides with complex patterns of posttranslational modifications. Mol. Cell. Proteomics 2010, 9 (5), 804-10.

111 127. Elias, J. E.; Gygi, S. P., Target-decoy search strategy for increased confidence in large- scale protein identifications by mass spectrometry. Nat. Methods 2007, 4 (3), 207-14. 128. Chalkley, R. J.; Baker, P. R.; Hansen, K. C.; Medzihradszky, K. F.; Allen, N. P.; Rexach, M.; Burlingame, A. L., Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of- flight mass spectrometer: I. How much of the data is theoretically interpretable by search engines? Mol. Cell. Proteomics 2005, 4 (8), 1189-93. 129. Csordas, A.; Reisinger, F.; Lavidas, I.; Dianes, J. A.; del-Toro, N.; Wang, R.; Ternent, T.; Perez-Riverol, Y.; Vizcaíno, J. A.; Griss, J.; Mayer, G.; Xu, Q.-W.; Hermjakob, H., 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 2015, 44 (D1), D447-D456. 130. Schrödinger Release 2018-1: Maestro, Schrödinger, LLC: New York, NY, 2018, 2016. 131. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A. V.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J. Gaussian 16 Rev. B.01, Wallingford, CT, 2016. 132. Grimme, S.; Ehrlich, S.; Goerigk, L., Effect of the damping function in dispersion corrected density functional theory. J. Comput. Chem. 2011, 32 (7), 1456-1465. 133. Cancès, E.; Mennucci, B.; Tomasi, J., A new integral equation formalism for the polarizable continuum model: Theoretical background and applications to isotropic and anisotropic dielectrics. J. Chem. Phys. 1997, 107 (8), 3032-3041. 134. Hanlon, R. T.; Messenger, J. B., Adaptive coloration in young cutttlefish (Sepia officinalis L.): The morphology and development of body patterns and their relation to behaviour. Philos. Trans. Royal Soc. B 1988, 320 (1200), 437-487. 135. Nijhout, H. F., Ommochrome pigmentation of the linea and rosa seasonal forms of Precis coenia (Lepidoptera: Nymphalidae). Arch. Insect Biochem. 1997, 36 (3), 215-222. 136. Vukusic, P.; Sambles, J. R., Photonic structures in biology. Nature 2003, 424 (6950), 852-855. 137. Li, L.; Kolle, S.; Weaver, J. C.; Ortiz, C.; Aizenberg, J.; Kolle, M., A highly conspicuous mineralized composite photonic architecture in the translucent shell of the blue-rayed limpet. Nat. Commun. 2015, 6, 6322. 138. Mäthger, L. M.; Hanlon, R. T., Anatomical basis for camouflaged polarized light communication in squid. Biol. Lett. 2006, 2, 494-496. 139. Kramer, R. M.; Crookes-Goodson, W. J.; Naik, R. R., The self-organizing properties of squid reflectin protein. Nat. Mater. 2007, 6 (7), 533-538. 140. Ordinario, D. D.; Phan, L.; Walkup, W. G.; Jocson, J.-M.; Karshalev, E.; Huesken, N.; Gorodetsky, A. A., Bulk protonic conductivity in a cephalopod structural protein. Nat. Chem. 2014, 6 (7), 597-603.

112 141. Sutherland, R. L.; Mäthger, L. M.; Hanlon, R. T.; Urbas, A. M.; Stone, M. O., Cephalopod coloration model. II. Multiple layer skin effects. J. Opt. Soc. Am. 2008, 25 (8), 2044-2054. 142. DeMartini, D. G.; Izumi, M.; Weaver, A. T.; Pandolfi, E.; Morse, D. E., Structures, Organization, and Function of Reflectin Proteins in Dynamically Tunable Reflective Cells. J. Biol. Chem. 2015, 290 (24), 15238-15249. 143. Florey, E., Ultrastructure and function of cephalopod chromatophores. Am. Zool. 1969, 9 (2), 429-442. 144. Florey, E.; Kriebel, M. E., Electrical and mechanical responses of chromatophore muscle fibers of squid, Loligo opalescens, to nerve stimulation and drugs. Z. Vgl. Physiol. 1969, 65 (1), 98-130. 145. Morin, S. A.; Shepherd, R. F.; Kwok, S. W.; Stokes, A. A.; Nemiroski, A.; Whitesides, G. M., Camouflage and Display for Soft Machines. Science 2012, 337 (6096), 828-832. 146. Yu, C. J.; Li, Y. H.; Zhang, X.; Huang, X.; Malyarchuk, V.; Wang, S. D.; Shi, Y.; Gao, L.; Su, Y. W.; Zhang, Y. H.; Xu, H. X.; Hanlon, R. T.; Huang, Y. G.; Rogers, J. A., Adaptive optoelectronic camouflage systems with designs inspired by cephalopod skins. Proc. Natl. Acad. Sci. U.S.A. 2014, 111 (36), 12998-13003. 147. Wang, Q.; Gossweiler, G. R.; Craig, S. L.; Zhao, X., Cephalopod-inspired design of electro-mechano-chemically responsive elastomers for on-demand fluorescent patterning. Nat. Commun. 2014, 5, 4899. 148. Chou, H.-H.; Nguyen, A.; Chortos, A.; To, J. W. F.; Lu, C.; Mei, J.; Kurosawa, T.; Bae, W.-G.; Tok, J. B. H.; Bao, Z., A chameleon-inspired stretchable electronic skin with interactive colour changing controlled by tactile sensing. Nat. Commun. 2015, 6, 8011. 149. Aubourg, S. P.; Torres-Arreola, W.; Trigo, M.; Ezquerra-Brauer, J. M., Partial characterization of jumbo squid skin pigment extract and its antioxidant potential in a marine oil system. Eur. J. Lipid Sci. Technol. 2016, 118, 1293-1304. 150. Elofsson, R.; Kauri, T., Ultrastructure of chromatophores of crangon and pandalus (crustacea). J. Ultrastruct. Res. 1971, 36 (1-2), 263-270. 151. Hase, S.; Wakamatsu, K.; Fujimoto, K.; Inaba, A.; Kobayashi, K.; Matsumoto, M.; Hoshi, M.; Negishi, S., Characterization of the pigment produced by the planarian, Dugesia ryukyuensis. Pigment Cell Res. 2006, 19 (3), 248-249. 152. Coates, B. S.; Hellmich, R. L.; Lewis, L. C., Two differentially expressed ommochrome- binding protein-like genes (obp1 and obp2) in larval fat body of the European corn borer, Ostrinia nubilalis. J. Insect Sci. 2005, 5 (1), 19. 153. Dontsov, A. E.; Fedorovich, I. B.; Lindström, M.; Ostrovsky, M. A., Comparative study of spectral and antioxidant properties of pigments from the eyes of two Mysis relicta (Crustacea, Mysidacea) populations, with different light damage resistance. J. Comp. Phys. B 1999, 169, 157-164. 154. Seligy, V. L., Ommochrome Pigments in Spiders. Comp. Biochem. Physiol., Part A Mol. Integr. Physiol. 1972, 42 (3), 699-709. 155. Kumar, A.; Williams, T. L.; Martin, C. A.; Figueroa-Navedo, A. M.; Deravi, L. F., Xanthommatin-Based Electrochromic Displays Inspired by Nature. ACS Appl Mater Interfaces 2018, 10 (49), 43177-43183. 156. Elgrishi, N.; Rountree, K. J.; McCarthy, B. D.; Rountree, E. S.; Eisenhart, T. T.; Dempsey, J. L., A Practical Beginner’s Guide to Cyclic Voltammetry. J. Chem. Educ. 2018, 95 (2), 197-206.

113 157. Levenson, R.; Bracken, C.; Bush, N.; Morse, D. E., Cyclable Condensation and Hierarchical Assembly of Metastable Reflectin Proteins, the Drivers of Tunable Biophotonics. J. Biol. Chem. 2015, 251 (8), 4058-4068. 158. Andouche, A.; Bassaglia, Y.; Baratte, S.; Bonnaud, L., Reflectin genes and development of iridophore patterns in Sepia officinalis embryos (Mollusca, Cephalopoda). Dev. Dyn. 2013, 242 (5), 560-571. 159. Bell, G. R. R.; Kuzirian, A. M.; Senft, S. L.; Mäthger, L. M.; Wardill, T. J.; Hanlon, R. T., Chromatophore radial muscle fibers anchor in flexible squid skin. Invertebr. Biol. 2013, 132 (2), 120-132. 160. Kumar, A.; Osgood, R. M.; Dinneen, S. R.; Koker, B. D.; Pang, R.; Deravi, L. F., Natural Light‐Scattering Nanoparticles Enable Visible through Short‐Wave Infrared Color Modulation. Adv. Opt. Mater. 2018, 6 (8), 1701369. 161. Kingston, A. C. N.; Kuzirian, A. M.; Hanlon, R. T.; Cronin, T. W., Visual phototransduction components in cephalopod chromatophores suggest dermal photoreception. J. Exp. Biol. 2015, 218 (10), 1596-1602. 162. Ramirez, M. D.; Oakley, T. H., Eye-independent, light-activated chromatophore expansion (LACE) and expression of phototransduction genes in the skin of Octopus bimaculoides. J. Exp. Biol. 2015, 218 (10), 1513-1520. 163. Pankey, M. S.; Minin, V. N.; Imholte, G. C.; Suchard, M. A.; Oakley, T. H., Predictable transcriptome evolution in the convergent and complex bioluminescent organs of squid. Proc. Natl. Acad. Sci. U.S.A. 2014, 111 (44), E4736-E4742. 164. Dennis, P. B.; Singh, K. M.; Vasudev, M. C.; Naik, R. R.; Crookes-Goodson, W. J., Research Update: A minimal region of squid reflectin for vapor-induced light scattering. APL Mater. 2017, 5 (12), 120701. 165. Delly, J. G. The Michel-Lévy Interference Color Chart – Microscopy’s Magical Color Key. https://www.mccrone.com/mm/the-michel-levy-interference-color-chart-microscopys- magical-color-key/ (accessed 6/7/18). 166. Brickner, D. G.; Ahmed, S.; Meldi, L.; Thompson, A.; Light, W.; Young, M.; Hickman, T. L.; Chu, F.; Fabre, E.; Brickner, J. H., Transcription factor binding to a DNA zip code controls interchromosomal clustering at the nuclear periphery. Dev. Cell. 2012, 22 (6), 1234-46. 167. Zybailov, B.; Coleman, M. K.; Florens, L.; Washburn, M. P., Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling. Anal. Chem. 2005, 77 (19), 6218- 24. 168. Rappsilber, J.; Ryder, U.; Lamond, A. I.; Mann, M., Large-scale proteomic analysis of the human spliceosome. Genome Res. 2002, 12 (8), 1231-45. 169. Ishihama, Y., Proteomic LC-MS systems using nanoscale liquid chromatography with tandem mass spectrometry. J. Chromatogr. A. 2005, 1067 (1-2), 73-83. 170. Choi, H.; Fermin, D.; Nesvizhskii, A. I., Significance Analysis of Spectral Count Data in Label-free Shotgun Proteomics. Mol. Cell. Proteomics 2008, 7 (12), 2373-2385. 171. Choi, H.; Kim, S.; Fermin, D.; Tsou, C.-C.; Nesvizhskii, A. I., QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics. J. Proteomics 2015, 129, 121-126. 172. Mirow, S., Skin color in the squids Loligo pealii and Loligo opalescens. I. Chromatophores. Z. Zellforsch. Mikrosk. Anat. 1972, 125 (2), 143-75.

114 173. Stavenga, D. G.; Leertouwer, H. L.; Wilts, B. D., Coloration principles of nymphaline butterflies – thin films, melanin, ommochromes and wing scale stacking. J. Exp. Biol. 2014, 217 (12), 2171-2180. 174. Hsiung, B.-K.; Justyn, N. M.; Blackledge, T. A.; Shawkey, M. D., Spiders have rich pigmentary and structural colour palettes. J. Exp. Biol. 2017, 220 (11), 1975-1983. 175. Saenko, S. V.; Teyssier, J.; van der Marel, D.; Milinkovitch, M. C., Precise colocalization of interacting structural and pigmentary elements generates extensive color pattern variation in Phelsuma lizards. BMC Biol. 2013, 11, 105. 176. Teyssier, J.; Saenko, S. V.; van der Marel, D.; Milinkovitch, M. C., Photonic crystals cause active colour change in chameleons. Nat. Commun. 2015, 6, 6368. 177. Williams, T., L.; Senft, S., L.; Yeo, J.; Martín-Martínez, F., J.; Kuzirian, A., M.; Martin, C., A.; DiBona, C., W.; Chen, C.-T.; Dinneen, S., R.; Nguyen, H., T.; Gomes, C., M.; Rosenthal, J., J.C.; MacManes, M., D.; Chu, F.; Buehler, M., J.; Hanlon, R., T.; Deravi, L., F., Dynamic pigmentary and structural coloration within cephalopod chromatophore organs. Nat. Commun. 2019, 10, 1004. 178. Guokui, Q.; B., D. P.; Yuji, Z.; Xiao, H.; E., B. J.; Zhongyuan, S.; J., C. G. W.; R., N. R.; G., O. F.; L., K. D., Recombinant reflectin‐based optical materials. J. Polym. Sci. B 2013, 51 (4), 254-264. 179. Florey, E.; Kriebel, M. E., Electrical and mechanical responses of chromatophore muscle fibers of the squid, Loligo opalescens, to nerve stimulation and drugs. Z. Vgl. Physiol. 1969, 65 (1), 98-130. 180. González Esquivel, D.; Ramírez-Ortega, D.; Pineda, B.; Castro, N.; Ríos, C.; Pérez de la Cruz, V., Kynurenine pathway metabolites and enzymes involved in redox reactions. Neuropharmacology 2017, 112, 331-345. 181. Dey, S. K.; Mukherjee, A., Catechol oxidase and phenoxazinone synthase: Biomimetic functional models and mechanistic studies. Coord. Chem. Rev. 2016, 310, 80-115. 182. Iwahashi, H.; Ishii, T., Detection of the oxidative products of 3-hydroxykynurenine using high-performance liquid chromatography electrochemical detection ultraviolet absorption detection electron spin resonance spectrometry and high-performance liquid chromatography electrochemical detection ultraviolet absorption detection mass spectrometry. J. Chromatogr. A 1997, 773 (1-2), 23-31. 183. Friedrich, J. M.; Ponce-de-León, C.; Reade, G. W.; Walsh, F. C., Reticulated vitreous carbon as an electrode material. J. Electroanal. Chem. 2004, 561, 203-217. 184. Alfonso-Súarez, P.; Kolliopoulos, A. V.; Smith, J. P.; Banks, C. E.; Jones, A. M., An experimentalist’s guide to electrosynthesis: the Shono oxidation. Tetrahedron Lett. 2015, 56 (49), 6863-6867. 185. Barry, C. E.; Nayar, P. G.; Begley, T. P., Phenoxazinone synthase: mechanism for the formation of the phenoxazinone chromophore of actinomycin. Biochemistry 1989, 28 (15), 6323- 6333. 186. Ishii, T.; Iwahashi, H.; Sugata, R.; Kido, R., Formation of hydroxanthommatin-derived radical in the oxidation of 3-hydroxykynurenine. Arch. Biochem. Biophys. 1992, 294 (2), 616- 622. 187. Ahlberg, E.; Falkenberg, F.; Manzanares, J. A.; Schiffrin, D. J., Convective mass transfer to partially recessed and porous electrodes. J. Electroanal. Chem. 2003, 548, 85-94.

115 188. Freeman, J. C.; Nayar, P. G.; Begley, T. P.; Villafranca, J. J., Stoichiometry and spectroscopic identity of copper centers in phenoxazinone synthase: A new addition to the blue copper oxidase family. Biochemistry 1993, 32 (18), 4826-4830. 189. Horváth, T.; Kaizer, J.; Speier, G., Functional phenoxazinone synthase models: Kinetic studies on the copper-catalyzed oxygenation of 2-aminophenol. J. Mol. Catal. A: Chem. 2004, 215 (1), 9-15. 190. Nematollahi, D.; Golabi, S. M., Electrochemical study of catechol and 4-methylcatechol in methanol. Application to the electro-organic synthesis of 4,5-dimethoxy-and 4-methoxy-5- methyl-o-benzoquinone. J. Electroanal. Chem. 1996, 405 (1), 133-140. 191. Nematollahi, D.; Shayani-jam, H., Kinetic Study of Electrochemically Induced Michael Reactions of o-Quinones with Meldrum’s Acid Derivatives. Synthesis of Highly Oxygenated Catechols. J. Org. Chem. 2008, 73 (9), 3428-3434. 192. Nematollahi, D.; Dehdashtian, S., Electrochemical oxidation of catechol in the presence of indole: a facile and one-pot method for the synthesis of trisindolyl-o-benzoquinone. Tetrahedron Lett. 2008, 49 (4), 645-649. 193. Nematollahi, D.; Shayani-Jam, H.; Alimoradi, M.; Niroomand, S., Electrochemical oxidation of acetaminophen in aqueous solutions: Kinetic evaluation of hydrolysis, hydroxylation and dimerization processes. Electrochim. Acta 2009, 54 (28), 7407-7415. 194. Esmaili, R.; Nematollahi, D., Electrochemical oxidation of 4-morpholinoaniline in aqueous solutions: Synthesis of a new trimer of 4-morpholinoaniline. Electrochim. Acta 2011, 56 (11), 3899-3904. 195. Shamsipur, M.; Davarani, S. S. H.; Nematollahi, D.; Taherpour, A.; Dehdashtian, S.; Pashabadi, A., One-step electrochemically driven production of aza macrocycle-based pseudo- cryptand: An accessible route for creating of diverse cryptand-resembles compounds. Electrochim. Acta 2018. 196. Yadav, A.; Mathur, P., p-Quinoneimine as an intermediate in the oxidative coupling of 2- amino-5-methylphenol to 4a,7-dimethyldihydro-2-aminophenoxazinone catalyzed by a monomeric copper(II) complex. Catal. Commun. 2014, 55, 1-5. 197. Bolognese, A.; Bonomo, R. P.; Chillemi, R.; Sciuto, S., Oxidation of 3- hydroxykynurenine. An EPR investigation. J. Heterocycl. Chem. 1990, 27 (7), 2207-2208.

116

Appendices

Table A1. Peptide count comparison of proteins identified from whole cell LC/MS/MS analysis. Peptide counts were normalized by predicted peptide count and by total peptide abundance per chromatophore type. (N =1 MS/MS for each color, which included a total of ~700 yellow, ~700 red, and ~1000 brown chromatophore organs that were collected and pooled from five total squid. Yellow chromatophores were pooled from two squid; Red chromatophores were pooled from two squid; and browns were pooled from three.)

Accession # Protein Name Category Brown Red Yellow

11724 Omega-crystallin Crystallin 256 170 290 6955 Elongation factor 1-alpha Metabolism 100 87 141

PREDICTED: smoothelin-like 12391 Protein-protein 52 83 60 protein 1 [Octopus bimaculoides]

PREDICTED: uncharacterized 11708 protein LOC106868335 [Octopus Uncharacterized 18 46 25 bimaculoides]

Extracellular 21216 Collagen alpha-4(VI) chain 54 28 74 Matrix

S-crystallin SL20-1-like [Octopus 13220 Crystallin 144 101 26 bimaculoides]

Deoxyribonuclease gamma [Jaculus 10569 Metabolism 66 63 39 jaculus]

Annexin A13-like isoform X2 8187 Protein-protein 76 55 39 [Octopus bimaculoides]

Lethal(2) giant larvae protein 8052 homolog 1-like [Octopus Cytoskeleton 13 38 9 bimaculoides]

6946 Glutathione S-tranferase mu Metabolism 69 64 69

Reflectin-like protein A2 5845 Reflectin 35 58 83 [Doryteuthis pealeii]

117 Myosin heavy chain isoform B 9491 Cytoskeleton 2 0 20 [Doryteuthis pealeii]

Reflectin-like protein A1 3328 Reflectin 26 38 44 [Doryteuthis pealeii]

PREDICTED: uncharacterized 11887 protein LOC106876157 [Octopus Uncharacterized 72 69 22 bimaculoides]

Reflectin-like protein B1 6590 Reflectin 31 44 76 [Doryteuthis pealeii]

6997 Melanotransferrin 4 Membrane 19 7 31 8179 Uniref: A0A0L8GHU8 Metabolism 38 50 9

PREDICTED: spectrin alpha chain- 9224 like isoform X3 [Octopus Cytoskeleton 8 1 10 bimaculoides]

Hypothetical protein 9342 LOTGIDRAFT_217535 [Lottia Uncharacterized 37 44 0 gigantea]

3200 Mitochondrial carrier (TC 2.A.29) Membrane 20 40 64

Glyceraldehyde-3-phosphate 3070 dehydrogenase-like isoform X1 Metabolism 45 63 11 [Octopus bimaculoides]

9086 Spectrin beta chain Cytoskeleton 13 5 16

Sulfotransferase family cytosolic 1B 7174 member 1-like [Octopus Metabolism 41 61 17 bimaculoides]

6812 Immunoglobulin E-set Metabolism 4 20 34 20952 ATP synthase subunit alpha Metabolism 15 12 23

356 Histone H4 [Larimichthys crocea] Nucleosome 96 52 122

PREDICTED: spectrin beta chain 9087 Cytoskeleton 7 2 9 isoform X5 [Crassostrea gigas]

Na+/K+ ATPase alpha subunit 3475 Ion Transport 7 7 18 [Doryteuthis pealeii]

118 13200 Annexin Protein-protein 21 15 31

PREDICTED: ATP synthase subunit 16139 beta, mitochondrial-like [Octopus Metabolism 14 15 28 bimaculoides]

PREDICTED: smoothelin-like 12158 Protein-protein 6 4 5 protein 1 [Octopus bimaculoides]

17802 Tubulin beta-4B chain Cytoskeleton 32 16 23 13974 Histone H2B Nucleosome 66 28 78 9250 TUBA1B Cytoskeleton 31 12 29

2093 Elongation factor 1-alpha Metabolism 17 15 23

Adenosylhomocysteinase [Astyanax 7273 Metabolism 25 29 0 mexicanus]

Omega-crystallin [Enteroctopus 9779 Crystallin 17 7 5 dofleini]

Voltage-dependent anion-selective 5905 Ion Transport 24 22 44 channel protein

PREDICTED: 14-3-3 protein 7408 Protein-protein 28 20 28 epsilon-like [Octopus bimaculoides]

PREDICTED: heat shock 70 kDa 2223 protein cognate 4 [Biomphalaria Metabolism 16 11 8 glabrata]

Basement membrane-specific Extracellular 13189 heparan sulfate proteoglycan core 1 3 3 Matrix protein

Tyrosinase-like [Octopus 19991 Metabolism 31 33 13 bimaculoides]

PREDICTED: protein disulfide- 8129 isomerase 2-like [Octopus Metabolism 12 6 15 bimaculoides]

Neutral and basic amino acid 10842 transport protein rBAT-like Membrane 12 7 10 [Octopus bimaculoides]

119 3110 Arginine kinase [Sepia pharaonis] Metabolism 22 20 7

Gelsolin-like protein 2 [Octopus 3163 Cytoskeleton 19 11 17 bimaculoides]

Spectrin alpha chain-like isoform X4 9230 Cytoskeleton 14 4 18 [Octopus bimaculoides]

Non-muscle myosin II heavy chain 10990 Cytoskeleton 4 3 1 [Doryteuthis pealeii]

Uncharacterized protein 12062 LOC109462197 isoform X3 Uncharacterized 26 29 4 [Branchiostoma belcheri]

Hypothetical protein 12758 OCBIM_22013362mg [Octopus Uncharacterized 5 2 1 bimaculoides]

Annexin A4-like [Octopus 6460 Protein-protein 14 15 16 bimaculoides]

Peroxiredoxin-1-like [Octopus 7073 Metabolism 31 25 19 bimaculoides]

PREDICTED: retinal dehydrogenase 3624 Metabolism 14 11 17 1-like [Octopus bimaculoides]

PREDICTED: glutathione S- 9264 transferase A-like [Octopus Metabolism 25 25 8 bimaculoides]

PREDICTED: sarcoplasmic/endoplasmic reticulum 11381 Ion Transport 3 2 14 calcium ATPase 1-like [Octopus bimaculoides]

7836 Paramyosin Cytoskeleton 2 0 11

Uncharacterized protein 8583 LOC106879019 [Octopus Uncharacterized 21 25 14 bimaculoides]

Extracellular 13274 Laminin subunit gamma-1 2 3 4 Matrix

120 Lysosomal aspartic protease-like 3360 Metabolism 16 17 9 isoform X2 [Octopus bimaculoides]

7611 Polyubiquitin-C [Chelonia mydas] Metabolism 26 29 37 11325 Tetraspanin Membrane 38 41 31

PREDICTED: histone H2A 2246 Nucleosome 45 25 57 [Octopus bimaculoides]

PREDICTED: transketolase-like 9625 protein 2 isoform X1 [Crassostrea Metabolism 8 15 0 gigas]

PREDICTED: hemocyte protein- glutamine gamma- 2809 Protein-Protein 3 8 19 glutamyltransferase-like isoform X1 [Octopus bimaculoides]

Ras-related protein Rab-1A [Haliotis 6218 Metabolism 13 22 19 discus discus]

PREDICTED: laminin subunit Extracellular 13237 alpha-like isoform X2 [Lingula 0 3 1 Matrix anatina]

S-crystallin SL20-1-like [Octopus 13224 Crystallin 20 15 3 bimaculoides]

PREDICTED: ras-related protein 3081 Metabolism 17 22 3 Rab-32-like [Octopus bimaculoides]

9551 Belongs to the 3-beta-HSD family Metabolism 9 8 3

Sulfotransferase family cytosolic 1B 7400 member 1-like [Octopus Metabolism 2 7 0 bimaculoides]

PREDICTED: hemocyte protein- glutamine gamma- 16456 Protein-protein 2 9 16 glutamyltransferase-like isoform X2 [Octopus bimaculoides]

Calpain-B-like isoform X17 11257 Metabolism 6 4 3 [Crassostrea virginica]

8660 Calcium ion binding Metabolism 1 6 2

121 PREDICTED: gelsolin-like protein 2 12464 Cytoskeleton 15 11 10 [Octopus bimaculoides]

2357 Enolase [Doryteuthis pealeii] Metabolism 8 7 5

Uncharacterized protein 6430 LOC106874846 isoform X3 Uncharacterized 5 11 0 [Octopus bimaculoides]

7486 Histone H3 [Octopus bimaculoides] Nucleosome 24 14 31

Mitochondrial substrate/solute 5134 Membrane 4 9 12 carrier

Membrane-associated protein Hem- 9016 like isoform X4 [Octopus Membrane 0 7 1 bimaculoides]

Annexin A4-like [Crassostrea 9583 Protein-Protein 10 8 3 virginica]

PREDICTED: alpha-adducin-like 10856 Protein-Protein 5 4 4 isoform X2 [Octopus bimaculoides]

PREDICTED: ras-like GTP-binding 11850 protein RHO isoform X2 [Octopus Metabolism 19 12 14 bimaculoides]

Uncharacterized protein 17820 LOC106873057 [Octopus Uncharacterized 20 14 9 bimaculoides]

PREDICTED: retinal dehydrogenase 3933 Metabolism 5 12 0 1-like [Octopus bimaculoides]

8042 Malate dehydrogenase Metabolism 10 9 6

PREDICTED: annexin A4-like 9156 Protein-Protein 9 9 4 [Octopus bimaculoides]

PREDICTED: citrate synthase, 13396 mitochondrial-like [Octopus Metabolism 9 7 3 bimaculoides]

Hypothetical protein 5071 BCR42DRAFT_419820 [Absidia Uncharacterized 8 8 10 repens]

122 PREDICTED: flotillin-2-like 7013 Membrane 2 3 11 [Octopus bimaculoides]

PREDICTED: fructose-bisphosphate 10059 aldolase-like [Octopus Metabolism 9 9 6 bimaculoides]

Hypothetical protein 11060 LOTGIDRAFT_227290 [Lottia Uncharacterized 7 11 11 gigantea]

PREDICTED: gelsolin-like protein 2 12437 Cytoskeleton 9 11 0 [Octopus bimaculoides]

ADP-ribosylation factor 1-like 19218 Metabolism 11 11 13 [Limulus polyphemus]

789 Histone H4 [Larimichthys crocea] Nucleosome 7 5 14

Annexin A4-like [Octopus 6642 Protein-protein 10 3 3 bimaculoides]

PREDICTED: transaldolase-like 9775 Metabolism 8 10 1 [Octopus bimaculoides]

Uncharacterized protein 11178 LOC106873394 [Octopus Uncharacterized 8 14 8 bimaculoides]

Hypothetical protein 13722 OCBIM_22013356mg [Octopus Uncharacterized 19 12 5 bimaculoides]

PREDICTED: puromycin-sensitive 19621 aminopeptidase-like [Octopus Metabolism 1 1 5 bimaculoides]

PREDICTED: ras-like protein 3 2610 Metabolism 12 14 2 isoform X1 [Octopus bimaculoides]

Peroxiredoxin 6 protein [Sepiella 6168 Metabolism 11 9 5 maindroni]

PREDICTED: uncharacterized 12463 protein LOC106880511 isoform X2 Uncharacterized 9 11 0 [Octopus bimaculoides]

123 Hypothetical protein 12766 OCBIM_22013362mg [Octopus Uncharacterized 2 2 0 bimaculoides]

Retrograde protein of 51 kDa 16407 Cytoskeleton 11 7 33 [Biomphalaria glabrata]

PREDICTED: cdc42 homolog 4018 Metabolism 15 12 12 [Octopus bimaculoides]

Lipase maturation factor 2-like 6452 Metabolism 2 9 3 isoform X2 [Octopus bimaculoides]

PREDICTED: calpain-11-like 7494 Metabolism 1 0 7 [Octopus bimaculoides]

PREDICTED: moesin-like [Octopus 8301 Protein-Protein 3 3 3 bimaculoides]

Cystathionine gamma-lyase 10800 Metabolism 8 7 0 [Crassostrea gigas]

PREDICTED: dolichyl- diphosphooligosaccharide--protein 11319 Metabolism 3 5 5 glycosyltransferase subunit 2-like [Octopus bimaculoides]

Hypothetical protein 17423 LOTGIDRAFT_223383 [Lottia Uncharacterized 14 10 2 gigantea]

PREDICTED: receptor expression- 19611 enhancing protein 5-like isoform X2 Membrane 22 18 7 [Octopus bimaculoides]

Tyrosine 3- monooxygenase/tryptophan 5- 61 Metabolism 6 5 8 monooxygenase activation protein, epsilon polypeptide 2

PREDICTED: 78 kDa glucose- 4205 regulated protein-like [Octopus Metabolism 3 2 4 bimaculoides]

peptidyl prolyl cis-trans isomerase A 5534 Metabolism 19 14 0 [Conus frigidus]

124 PREDICTED: uncharacterized 6866 protein LOC106872046 [Octopus Uncharacterized 8 7 1 bimaculoides]

PREDICTED: laminin subunit beta- Extracellular 9620 0 2 1 1-like [Octopus bimaculoides] Matrix

PREDICTED: flotillin-1-like 3161 Membrane 0 2 8 isoform X1 [Aplysia californica]

nucleoredoxin-like protein 2 5970 Metabolism 9 9 15 [Acanthaster planci]

PREDICTED: pantetheinase-like 6668 Metabolism 2 2 7 [Octopus bimaculoides]

PREDICTED: extended 7421 synaptotagmin-2-like isoform X1 Membrane 4 2 1 [Octopus bimaculoides]

PREDICTED: uncharacterized 9133 protein LOC106876157 [Octopus Uncharacterized 15 0 12 bimaculoides]

PREDICTED: rab GDP dissociation 9154 inhibitor alpha-like [Octopus Metabolism 2 8 2 bimaculoides]

PREDICTED: NADP-dependent 10684 malic enzyme-like isoform X1 Metabolism 3 6 0 [Octopus bimaculoides]

Clathrin heavy chain 1 isoform X2 12448 Membrane 1 1 1 [Mizuhopecten yessoensis]

PREDICTED: cytoplasmic dynein 1 13024 heavy chain 1 isoform X12 Metabolism 0 0 0 [Crassostrea gigas]

PREDICTED: cartilage matrix Extracellular 13169 11 6 14 protein-like [Octopus bimaculoides] Matrix

Hypothetical protein 13720 OCBIM_22013360mg [Octopus Uncharacterized 22 12 0 bimaculoides]

125 PREDICTED: kynurenine 17326 formamidase [Chrysochloris Metabolism 7 19 0 asiatica]

Hypothetical protein A1O3_08667 19928 Uncharacterized 4 8 10 [Capronia epimyces CBS 606.96]

PREDICTED: guanine nucleotide- 1839 binding protein G(o) subunit alpha Metabolism 4 5 4 isoform X1 [Octopus bimaculoides]

Vacuolar protein sorting-associated 6813 Metabolism 4 6 10 protein 4B

PREDICTED: transitional 7691 endoplasmic reticulum ATPase-like Metabolism 2 2 1 [Octopus bimaculoides]

PREDICTED: hephaestin-like 8860 Metabolism 3 1 2 protein [Octopus bimaculoides]

Reflectin-like protein A2 9165 Reflectin 7 14 14 [Doryteuthis pealeii]

Copine-3-like isoform X2 [Octopus 11316 Membrane 5 9 9 bimaculoides]

Collagen alpha-4(VI) chain-like Extracellular 13241 0 5 8 [Lingula anatina] Matrix

PREDICTED: ATP synthase subunit 19972 O, mitochondrial-like [Octopus Metabolism 5 5 7 bimaculoides]

PREDICTED: ATP synthase subunit 20271 g, mitochondrial-like [Octopus Metabolism 15 15 20 bimaculoides]

PREDICTED: FAS-associated 3253 Metabolism 3 5 1 factor 2-like [Octopus bimaculoides]

Reflectin-like protein A1 3329 Reflectin 5 9 7 [Doryteuthis opalescens]

126 PREDICTED: saccharopine 5952 dehydrogenase-like oxidoreductase Metabolism 4 4 4 isoform X1 [Pygocentrus nattereri]

Uncharacterized protein 6281 LOC106874846 isoform X3 Uncharacterized 12 19 4 [Octopus bimaculoides]

Membrane-associated protein Hem- 9052 like isoform X4 [Octopus Membrane 2 5 0 bimaculoides]

PREDICTED: LOW QUALITY PROTEIN: dolichyl- 9761 diphosphooligosaccharide--protein Metabolism 2 2 6 glycosyltransferase 48 kDa subunit- like [Octopus bimaculoides]

Syntenin-1-like [Octopus 10007 Protein-protein 6 12 0 bimaculoides]

PREDICTED: gelsolin-like protein 2 12583 Cytoskeleton 4 5 3 [Octopus bimaculoides]

PREDICTED: nucleoside 17618 diphosphate kinase-like [Octopus Metabolism 8 8 8 bimaculoides]

PREDICTED: dolichyl- diphosphooligosaccharide--protein 3386 Metabolism 1 2 3 glycosyltransferase subunit 1-like [Octopus bimaculoides]

PREDICTED: serine/threonine- protein phosphatase 2A 65 kDa 4186 regulatory subunit A alpha isoform- Metabolism 2 4 1 like isoform X2 [Biomphalaria glabrata]

PREDICTED: uncharacterized 4645 protein LOC106884239 [Octopus Uncharacterized 5 3 0 bimaculoides]

5108 Beta-tubulin [Doryteuthis pealeii] Cytoskeleton 4 3 4

127 PREDICTED: protocadherin-11 X- 5136 linked-like isoform X6 [Octopus Membrane 4 7 0 bimaculoides]

PREDICTED: alpha-actinin, 6907 sarcomeric-like isoform X1 Cytoskeleton 1 0 2 [Octopus bimaculoides]

7007 Elongation factor 1-alpha Metabolism 3 5 0

PREDICTED: V-type proton 7381 ATPase catalytic subunit A Ion Transport 2 5 0 [Octopus bimaculoides]

glutathione S-transferase 2 8039 Metabolism 7 12 0 [Idiosepius paradoxus]

PREDICTED: plasma membrane 8664 calcium-transporting ATPase 2-like Ion Transport 1 1 2 [Octopus bimaculoides]

6-phosphogluconate dehydrogenase, 8671 decarboxylating-like [Crassostrea Metabolism 2 6 0 virginica]

PREDICTED: aquaporin-like 9514 Membrane 9 0 15 [Octopus bimaculoides]

Peptidase C2, calpain, large subunit, 9844 Metabolism 1 3 1 domain III

Heat shock protein 90 [Octopus 9929 Metabolism 1 1 2 vulgaris]

Copine-3-like isoform X2 [Octopus 11230 Membrane 4 5 1 bimaculoides]

PREDICTED: ATP synthase subunit 16523 b, mitochondrial-like [Octopus Metabolism 3 6 3 bimaculoides]

PREDICTED: ras-related protein 19642 Metabolism 4 6 6 Rab-2 [Octopus bimaculoides]

PREDICTED: glucose-6-phosphate 3686 1-dehydrogenase-like [Octopus Metabolism 1 5 0 bimaculoides]

128 Hypothetical protein 5750 LOTGIDRAFT_137310 [Lottia Uncharacterized 6 8 0 gigantea]

PREDICTED: ras-related protein 7162 Metabolism 7 14 3 Rab-10-like [Aplysia californica]

PREDICTED: profilin-like 7265 Cytoskeleton 22 11 5 [Amphimedon queenslandica]

PREDICTED: uncharacterized 8371 protein LOC106870819 [Octopus Uncharacterized 5 2 10 bimaculoides]

8515 Isocitrate dehydrogenase [NADP] Metabolism 2 0 5

uncharacterized protein 8780 LOC106876168 [Octopus Uncharacterized 0 1 6 bimaculoides]

Extracellular 9781 Collagen, type IV 1 1 1 Matrix

PREDICTED: reticulon-1-A-like 11498 Membrane 10 7 7 isoform X2 [Octopus bimaculoides]

Reflectin-like protein A1 17742 Reflectin 4 7 15 [Doryteuthis pealeii]

PREDICTED: prosaposin-like 3203 Metabolism 2 1 1 isoform X2 [Octopus bimaculoides]

Hypothetical protein 3763 OCBIM_22016971mg [Octopus Uncharacterized 20 7 13 bimaculoides]

PREDICTED: protocadherin-11 X- 5585 linked-like isoform X6 [Octopus Membrane 4 6 2 bimaculoides]

Hypothetical protein 8144 LOTGIDRAFT_231723 [Lottia Uncharacterized 1 3 0 gigantea]

PREDICTED: aspartate 8255 aminotransferase, mitochondrial-like Metabolism 2 0 4 [Octopus bimaculoides]

129 Transmembrane emp24 domain- 8594 containing protein 10-like Protein-Protein 4 4 4 [Mizuhopecten yessoensis]

Uncharacterized protein 8828 LOC106879019 [Octopus Uncharacterized 1 2 1 bimaculoides]

PREDICTED: von Willebrand factor 9592 A domain-containing protein 5A- Uncharacterized 2 2 0 like [Octopus bimaculoides]

10106 Vitellinogen, open beta-sheet Metabolism 0 0 1

PREDICTED: vinculin-like 10692 Cytoskeleton 1 1 1 [Octopus bimaculoides]

PREDICTED: smoothelin-like 12147 Protein-protein 6 3 9 protein 1 [Octopus bimaculoides]

PREDICTED: neural cell adhesion 12975 molecule 2-like [Octopus Membrane 0 5 0 bimaculoides]

PREDICTED: putative ATP 21163 synthase subunit f, mitochondrial Membrane 6 6 6 [Octopus bimaculoides]

450 Gq-alpha [Doryteuthis pealeii] Metabolism 4 3 0

PREDICTED: putative ATP 521 synthase subunit f, mitochondrial Metabolism 7 3 7 [Octopus bimaculoides]

PREDICTED: ribosyldihydronicotinamide 565 Metabolism 2 10 0 dehydrogenase [quinone]-like [Octopus bimaculoides]

PREDICTED: zinc finger Ran- 1273 binding domain-containing protein Protein-Protein 2 2 5 2-like [Octopus bimaculoides]

PREDICTED: V-type proton 3265 ATPase 16 kDa proteolipid subunit Ion Transport 11 16 0 [Octopus bimaculoides]

130 PREDICTED: retinal dehydrogenase 3746 Metabolism 0 3 2 1-like [Octopus bimaculoides]

PREDICTED: glycerol-3-phosphate 4048 dehydrogenase, mitochondrial-like Metabolism 0 1 2 isoform X1 [Octopus bimaculoides]

Actin depolymerizing protein 4858 Metabolism 5 3 5 [Gonapodya prolifera JEL478]

PREDICTED: endoplasmin-like 6044 Metabolism 1 2 1 [Octopus bimaculoides]

PREDICTED: V-type proton 6347 ATPase subunit B [Octopus Ion Transport 1 4 0 bimaculoides]

PREDICTED: putative universal 6702 stress protein SAS1637 [Octopus Metabolism 12 8 0 bimaculoides]

Elongation factor Tu 7247 Metabolism 0 0 7 [Tenacibaculum dicentrarchi]

PREDICTED: stomatin-like protein 7384 2, mitochondrial [Octopus Membrane 4 3 0 bimaculoides]

PREDICTED: GTPase HRas 7407 Metabolism 8 5 0 isoform X2 [Octopus bimaculoides]

PREDICTED: probable 8509 phosphoglycerate mutase [Octopus Metabolism 1 5 0 bimaculoides]

Uncharacterized protein 8706 LOC106879019 [Octopus Uncharacterized 2 2 1 bimaculoides]

PREDICTED: retinol 8855 dehydrogenase 12-like [Octopus Metabolism 3 0 4 bimaculoides]

PREDICTED: zinc 9091 metalloproteinase nas-15-like Metabolism 2 0 1 [Aplysia californica]

131 Talin-1-like isoform X21 10030 Cytoskeleton 0 0 0 [Crassostrea virginica]

PREDICTED: dolichyl- diphosphooligosaccharide--protein 10086 Metabolism 1 1 3 glycosyltransferase subunit STT3A isoform X1 [Octopus bimaculoides]

Adenylyl cyclase-associated protein 10557 1-like isoform X3 [Crassostrea Metabolism 1 3 0 virginica]

PREDICTED: glutamate 11283 dehydrogenase, mitochondrial-like Metabolism 2 1 2 [Octopus bimaculoides]

PREDICTED: disco-interacting 11801 protein 2 homolog A-like [Octopus Membrane 0 0 2 bimaculoides]

PREDICTED: gelsolin-like protein 2 12681 Cytoskeleton 2 1 2 [Octopus bimaculoides]

PREDICTED: cytochrome b-c1 complex subunit 2, mitochondrial- 13083 Metabolism 0 3 4 like isoform X2 [Octopus bimaculoides]

PREDICTED: gelsolin-like protein 2 13126 Cytoskeleton 0 0 1 [Octopus bimaculoides]

Reflectin-like protein C1 16776 Reflectin 0 3 11 [Doryteuthis opalescens]

PREDICTED: ATP-dependent RNA 17985 helicase eIF4A-like [Octopus Nucleosome 2 3 0 bimaculoides]

PREDICTED: probable caffeoyl- 18701 CoA O-methyltransferase 1 Metabolism 4 3 0 [Octopus bimaculoides]

PREDICTED: excitatory amino acid 266 transporter 1-like [Octopus Ion Transport 2 2 5 bimaculoides]

132 PREDICTED: myosin catalytic light 3058 chain LC-1, mantle muscle-like Metabolism 3 0 9 [Octopus bimaculoides]

PREDICTED: phytanoyl-CoA 3062 dioxygenase, peroxisomal-like Metabolism 3 0 9 isoform X1 [Octopus bimaculoides]

PREDICTED: deoxynucleoside triphosphate triphosphohydrolase 3231 Metabolism 1 1 2 SAMHD1-like [Octopus bimaculoides]

PREDICTED: CD81 protein-like 3372 Membrane 8 8 0 isoform X1 [Octopus bimaculoides]

Hypothetical protein CGI_10001848 3962 Uncharacterized 0 0 7 [Crassostrea gigas]

PREDICTED: transmembrane 4948 protein 245-like [Octopus Membrane 1 3 0 bimaculoides]

Protein kinase C and casein kinase 5009 substrate in neurons protein 1-like Metabolism 7 7 0 [Octopus bimaculoides]

PREDICTED: NAD(P) 5888 transhydrogenase, mitochondrial- Metabolism 1 2 1 like [Aplysia californica]

6375 Defender against cell death 1 Membrane 13 0 13

PREDICTED: GTP-binding protein 7113 Metabolism 4 2 2 SAR1b-like [Octopus bimaculoides]

PREDICTED: malate 7276 dehydrogenase, cytoplasmic-like Metabolism 5 2 0 [Octopus bimaculoides]

PREDICTED: serine/threonine- 7430 protein phosphatase alpha-2 isoform Protein-Protein 3 3 0 isoform X3 [Octopus bimaculoides]

Cluster: Flagellar biosynthetic 8572 Cytoskeleton 3 0 9 protein FlhB

133 PREDICTED: GLIPR1-like protein 9441 Metabolism 1 1 2 2 [Octopus bimaculoides]

V-type proton ATPase 116 kDa 10045 subunit a-like isoform X2 [Limulus Ion Transport 2 2 0 polyphemus]

Low-density lipoprotein receptor- 11310 related protein 2-like [Parasteatoda Membrane 4 9 4 tepidariorum]

PREDICTED: catenin beta-like 11350 Protein-Protein 1 0 2 [Octopus bimaculoides]

PREDICTED: 40S ribosomal 16300 protein S4-like [Octopus Metabolism 4 1 0 bimaculoides]

PREDICTED: 60S ribosomal 18985 protein L14-like isoform X1 Metabolism 5 0 5 [Octopus bimaculoides]

Tyrosinase-like [Octopus 20501 Metabolism 7 3 3 bimaculoides]

PREDICTED: tryptophan 2,3- 425 Metabolism 2 5 0 dioxygenase-like [Lingula anatina]

Hypothetical protein 1801 OCBIM_22013360mg [Octopus Uncharacterized 7 4 0 bimaculoides]

Iduronate 2-sulfatase-like [Octopus 2347 Metabolism 3 2 0 bimaculoides]

PREDICTED: actin-related protein 2714 2/3 complex subunit 4 [Octopus Cytoskeleton 4 2 0 bimaculoides]

PREDICTED: putative 3066 aminopeptidase W07G4.4 [Octopus Metabolism 1 2 0 bimaculoides]

PREDICTED: pyruvate dehydrogenase E1 component 3384 Metabolism 1 2 0 subunit alpha, mitochondrial-like [Octopus bimaculoides]

134 PREDICTED: abhydrolase domain- 4418 containing protein 16A-like isoform Metabolism 1 1 1 X1 [Octopus bimaculoides]

Sodium-calcium exchanger 4636 Ion Transport 3 0 6 [Doryteuthis pealeii]

Translocon-associated protein 5143 Metabolism 2 0 5 subunit beta

Extracellular 5469 Matrilin 2 1 0 2 Matrix

PREDICTED: ADP-ribosylation 5721 factor-like protein 8B [Octopus Metabolism 2 2 2 bimaculoides]

PREDICTED: acyl-coenzyme A 5843 thioesterase 9, mitochondrial-like Metabolism 1 2 0 [Branchiostoma belcheri]

PREDICTED: collagen alpha-4(VI) Extracellular 6680 0 0 1 chain-like [Octopus bimaculoides] Matrix

PREDICTED: large neutral amino 7375 acids transporter small subunit 2-like Metabolism 2 2 2 isoform X1 [Octopus

Calmodulin [Mucor circinelloides f. 7942 Protein-Protein 5 0 3 circinelloides 1006PhL]

Annexin A13-like isoform X2 8360 Protein-protein 1 2 0 [Octopus bimaculoides]

PREDICTED: nicastrin-like isoform 8518 Metabolism 2 1 0 X1 [Octopus bimaculoides]

Thioester-containing protein 1 9076 Protein-Protein 0 0 1 [Euprymna scolopes]

9821 Prohibitin 2 [Sepiella japonica] Metabolism 1 1 1

PREDICTED: copine-3-like 10121 Membrane 1 0 2 [Octopus bimaculoides]

Histidine ammonia-lyase-like 10973 Metabolism 0 0 2 [Mizuhopecten yessoensis]

135 PREDICTED: integrin alpha pat-2- 11141 Membrane 1 0 0 like [Octopus bimaculoides]

PREDICTED: ornithine 11142 aminotransferase, mitochondrial-like Metabolism 1 3 0 isoform X1 [Octopus bimaculoides]

11219 CBN-EMB-9 protein Protein-protein 0 1 0

PREDICTED: reticulon-1-A isoform 11499 Membrane 9 9 9 X5 [Crassostrea gigas]

PREDICTED: reticulon-1-A-like 11502 Membrane 3 3 3 isoform X3 [Octopus bimaculoides]

PREDICTED: reticulon-1-A isoform 11503 Membrane 4 4 4 X9 [Crassostrea gigas]

Low-density lipoprotein receptor- 11655 related protein 2-like [Parasteatoda Membrane 1 2 0 tepidariorum]

NADH-cytochrome b5 reductase 3- 12660 like isoform X1 [Crassostrea Metabolism 2 2 2 virginica]

PREDICTED: copine-8-like 13273 Membrane 1 1 1 [Octopus bimaculoides]

PREDICTED: ganglioside GM2 16209 activator-like [Octopus Protein-protein 0 6 0 bimaculoides]

PREDICTED: focadhesin-like 16329 Membrane 8 0 16 [Octopus bimaculoides]

PREDICTED: ectonucleoside 16614 triphosphate diphosphohydrolase 1- Membrane 2 4 0 like [Octopus bimaculoides]

PREDICTED: uncharacterized 16835 protein LOC106877008 [Octopus Uncharacterized 4 4 4 bimaculoides]

136 PREDICTED: ATP synthase subunit 17881 d, mitochondrial-like [Octopus Metabolism 7 3 0 bimaculoides]

PREDICTED: adenosine deaminase- 17993 Metabolism 1 2 0 like [Octopus bimaculoides]

18123 Ferritin [Sepiella maindroni] Metabolism 3 5 0

Uncharacterized protein 18922 LOC106873057 [Octopus Uncharacterized 7 3 0 bimaculoides]

PREDICTED: synaptophysin-like 21123 Protein-Protein 6 3 0 isoform X1 [Octopus bimaculoides]

PREDICTED: transmembrane 335 emp24 domain-containing protein 2 Protein-Protein 5 0 0 [Crassostrea gigas]

PREDICTED: NADH 366 dehydrogenase [ubiquinone] 1 Metabolism 4 0 4 subunit C2 [Cephus cinctus]

PREDICTED: protein Mo25-like 672 Metabolism 1 1 0 [Octopus bimaculoides]

PREDICTED: mesoderm-specific 1078 transcript homolog protein-like Metabolism 6 6 0 [Octopus bimaculoides]

PREDICTED: protein deglycase DJ- 2064 1-like isoform X1 [Priapulus Metabolism 4 0 0 caudatus]

PREDICTED: elongation factor 1- 2454 Metabolism 0 4 0 gamma-like [Octopus bimaculoides]

PREDICTED: vesicular integral- 2573 membrane protein VIP36-like Protein-Protein 0 2 2 [Octopus bimaculoides]

PREDICTED: acylamino-acid- 3209 releasing enzyme-like [Octopus Metabolism 1 0 1 bimaculoides]

137 Chain A, Crystal Structure Analysis 4004 Of Neuronal Sec1 From The Squid Membrane 1 1 0 L. Pealei

PREDICTED: beta-hexosaminidase 4141 subunit beta-like [Octopus Metabolism 1 0 1 bimaculoides]

PREDICTED: actophorin-like 4191 Metabolism 3 3 0 [Octopus bimaculoides]

PREDICTED: 78 kDa glucose- 4200 regulated protein-like [Octopus Metabolism 2 0 2 bimaculoides]

PREDICTED: uncharacterized 4531 protein LOC106874881 [Octopus Uncharacterized 3 0 3 bimaculoides]

PREDICTED: calcium-binding mitochondrial carrier protein 4749 Ion Transport 0 2 0 SCaMC-2-like [Octopus bimaculoides]

5069 Uncharacterized Uncharacterized 5 5 0

PREDICTED: uncharacterized 5145 protein LOC106876157 [Octopus Uncharacterized 7 0 0 bimaculoides]

PREDICTED: ras-related protein 5445 Metabolism 2 2 0 Rab-5B-like [Octopus bimaculoides]

Low-density lipoprotein receptor- 5459 related protein 2-like [Parasteatoda Membrane 0 3 3 tepidariorum]

Low-density lipoprotein receptor- 5460 related protein 2-like [Parasteatoda Membrane 0 3 3 tepidariorum]

PREDICTED: cathepsin L1-like 6049 Metabolism 1 1 0 [Octopus bimaculoides]

138 PREDICTED: 26S protease 6051 regulatory subunit 6B [Octopus Metabolism 1 1 0 bimaculoides]

PREDICTED: 40S ribosomal 6453 Metabolism 2 0 2 protein S5 [Octopus bimaculoides]

Hypothetical protein 6508 OCBIM_22013360mg [Octopus Uncharacterized 5 0 0 bimaculoides]

PREDICTED: triosephosphate 6589 isomerase-like [Octopus Metabolism 2 2 0 bimaculoides]

PREDICTED: wiskott-Aldrich 6809 syndrome protein family member 3- Protein-Protein 0 4 0 like [Octopus bimaculoides]

PREDICTED: alpha-N- 6810 acetylgalactosaminidase-like Metabolism 2 2 0 [Octopus bimaculoides]

Ubiquitin-conjugating enzyme E2 7324 Metabolism 0 6 0 L3-like [Limulus polyphemus]

PREDICTED: T-cell 7522 immunomodulatory protein-like Protein-Protein 1 1 0 [Octopus bimaculoides]

PREDICTED: translocon-associated 7534 protein subunit gamma-like Metabolism 4 0 4 [Octopus bimaculoides]

PREDICTED: ubiquitin carboxyl- 7644 terminal hydrolase-like [Crassostrea Metabolism 2 2 0 gigas]

PREDICTED: glycogen 7681 phosphorylase, brain form-like Metabolism 1 1 0 [Octopus bimaculoides]

PREDICTED: aspartyl/asparaginyl 7725 beta-hydroxylase-like [Octopus Metabolism 5 0 0 bimaculoides]

139 PREDICTED: surfeit locus protein 7804 Membrane 2 0 2 4-like [Octopus bimaculoides]

PREDICTED: 2-oxoglutarate 7852 dehydrogenase, mitochondrial-like Metabolism 1 1 0 isoform X2 [Octopus bimaculoides]

PREDICTED: membrane-associated 7885 progesterone receptor component 1- Membrane 2 2 0 like [Octopus bimaculoides]

Hypothetical protein 8081 OCBIM_22013360mg [Octopus Uncharacterized 4 0 0 bimaculoides]

PREDICTED: lamin-B1-like 8804 Cytoskeleton 1 0 0 [Octopus bimaculoides]

9092 Spectrin beta chain Cytoskeleton 0 2 0

PREDICTED: retinol 9258 dehydrogenase 11-like isoform X2 Metabolism 1 0 1 [Octopus bimaculoides]

Hypothetical protein 9340 OCBIM_22013360mg [Octopus Uncharacterized 5 5 0 bimaculoides]

PREDICTED: acid ceramidase-like 9552 Metabolism 1 1 0 [Octopus bimaculoides]

PREDICTED: dihydropyrimidinase- 9689 Metabolism 0 2 0 like [Octopus bimaculoides]

PREDICTED: solute carrier family 2, facilitated glucose transporter 9951 Membrane 2 2 0 member 1-like [Octopus bimaculoides]

PREDICTED: 60 kDa heat shock 10429 protein, mitochondrial-like [Octopus Metabolism 1 1 0 bimaculoides]

140 PREDICTED: UDP- glucose:glycoprotein 10788 Metabolism 0 0 0 glucosyltransferase 1-like [Octopus bimaculoides]

Hypothetical protein 11366 OCBIM_22002744mg [Octopus Uncharacterized 0 1 1 bimaculoides]

Ribonuclease E [Pseudoalteromonas 11501 Metabolism 3 0 3 sp. PAMC 28425]

PREDICTED: basement membrane- specific heparan sulfate Extracellular 13113 0 3 3 proteoglycan core protein-like Matrix isoform X4 [Octopus bimaculoides]

Chain A, Diisopropyl 13761 Fluorophosphatase (Dfpase), D121e Metabolism 5 5 0 Mutant [Loligo vulgaris]

PREDICTED: sialin-like [Octopus 16191 Membrane 2 2 0 bimaculoides]

PREDICTED: 40S ribosomal 16374 protein S3-B-like [Octopus Metabolism 3 0 0 bimaculoides]

PREDICTED: erlin-1-like [Octopus 16868 Protein-Protein 4 0 0 bimaculoides]

PREDICTED: 60S ribosomal 17024 protein L13a-like [Octopus Metabolism 3 0 0 bimaculoides]

Similar to Drosophila melanogaster 17150 Metabolism 5 0 0 RpS3A [Drosophila yakuba]

Cysteine protease ATG4B-like 17604 Metabolism 2 2 0 [Crassostrea virginica]

PREDICTED: nucleoside 17672 diphosphate kinase-like [Octopus Metabolism 1 1 0 bimaculoides]

141 PREDICTED: AP-1 complex 17897 subunit beta-1-like isoform X3 Protein-Protein 1 1 0 [Octopus bimaculoides]

PREDICTED: 40S ribosomal 17900 protein S8-like [Octopus Metabolism 2 0 2 bimaculoides]

Copper zinc superoxide dismutase 17939 Metabolism 0 7 0 [Sepiella maindroni]

40S ribosomal protein S18 18329 Metabolism 2 0 2 [Exaiptasia pallida]

PREDICTED: hydroxysteroid 11- 19124 beta-dehydrogenase 1-like protein Metabolism 0 0 5 [Octopus bimaculoides]

PREDICTED: ras-related protein 19871 Metabolism 2 2 0 Rab-11A [Lingula anatina]

PREDICTED: calumenin-A-like 20007 Metabolism 2 0 0 [Octopus bimaculoides]

PREDICTED: 40S ribosomal 20325 Metabolism 4 0 0 protein S16 [Octopus bimaculoides]

PREDICTED: peptidyl-prolyl cis- 20347 trans isomerase B-like [Octopus Metabolism 2 0 2 bimaculoides]

PREDICTED: minor 98 histocompatibility antigen H13-like Membrane 0 0 2 isoform X1 [Octopus bimaculoides]

PREDICTED: mitochondrial carrier 131 homolog 2-like [Octopus Ion Transport 0 2 0 bimaculoides]

PREDICTED: pyruvate dehydrogenase E1 component 306 Metabolism 2 0 0 subunit beta, mitochondrial-like [Octopus bimaculoides]

PREDICTED: maestro heat-like 320 repeat-containing protein family Membrane 3 0 0 member 1 [Octopus bimaculoides]

142 PREDICTED: 60S ribosomal 362 protein L27-like [Octopus Metabolism 3 0 0 bimaculoides]

PREDICTED: LETM1 and EF-hand domain-containing protein 1, 641 Ion Transport 0 0 0 mitochondrial-like isoform X2 [Octopus bimaculoides]

PREDICTED: cleft lip and palate 696 transmembrane protein 1 homolog Metabolism 1 0 0 [Octopus bimaculoides]

PREDICTED: heparanase-like Extracellular 717 1 0 0 [Octopus bimaculoides] Matrix

PREDICTED: ras-related protein 985 Rab-18A-like isoform X2 [Octopus Metabolism 2 0 0 bimaculoides]

PREDICTED: erlin-1-like [Octopus 1037 Protein-Protein 6 0 0 bimaculoides]

Octopine dehydrogenase 1527 Metabolism 0 1 0 [Doryteuthis opalescens]

PREDICTED: digestive cysteine 1882 Metabolism 1 0 0 proteinase 2-like [Lingula anatina]

PREDICTED: mitochondrial 1989 pyruvate carrier 2-like [Octopus Ion Transport 0 0 4 bimaculoides]

PREDICTED: 60S ribosomal 2213 protein L15-like [Octopus Metabolism 2 0 0 bimaculoides]

PREDICTED: cytochrome c oxidase 2607 subunit 5B, mitochondrial-like Metabolism 3 0 0 [Octopus bimaculoides]

Translationally controlled tumor- 2727 associated [Rhizopus microsporus Protein-Protein 0 3 0 var. microsporus]

Glutathione-S-transferase [Haliotis 3393 Metabolism 2 0 0 madaka]

143 PREDICTED: signal recognition 3433 particle receptor subunit beta-like Membrane 2 0 0 [Octopus bimaculoides]

PREDICTED: T-complex protein 1 3567 subunit beta-like [Octopus Metabolism 1 0 0 bimaculoides]

PREDICTED: uncharacterized 3895 protein LOC106868120 [Octopus Uncharacterized 0 1 0 bimaculoides]

PREDICTED: uncharacterized 4211 protein LOC106882100 [Octopus Uncharacterized 1 0 0 bimaculoides]

PREDICTED: transmembrane 4247 emp24 domain-containing protein Protein-Protein 0 0 2 eca isoform X1 [Crassostrea gigas]

PREDICTED: glyceraldehyde-3- 4251 phosphate dehydrogenase-like Metabolism 0 0 1 isoform X1 [Octopus bimaculoides]

PREDICTED: succinyl-CoA ligase 4370 subunit alpha, mitochondrial-like Metabolism 0 0 2 [Octopus bimaculoides]

PREDICTED: uncharacterized threonine-rich GPI-anchored 4407 Uncharacterized 0 2 0 glycoprotein PJ4664.02-like [Octopus bimaculoides]

Hypothetical protein 4834 OCBIM_22030393mg [Octopus Uncharacterized 4 0 0 bimaculoides]

PREDICTED: acetolactate synthase- 4918 Metabolism 0 2 0 like protein [Octopus bimaculoides]

PREDICTED: 60S ribosomal 5046 protein L28-like [Octopus Metabolism 3 0 0 bimaculoides]

144 PREDICTED: trafficking protein 5056 particle complex subunit 12-like Protein-Protein 0 0 1 [Octopus bimaculoides]

PREDICTED: opioid growth factor 5325 receptor-like protein 1 isoform X2 Protein-Protein 2 0 0 [Octopus bimaculoides]

Hypothetical protein 5334 OCBIM_22013360mg [Octopus Uncharacterized 4 0 0 bimaculoides]

PREDICTED: uncharacterized 5396 protein LOC106883080 [Octopus Uncharacterized 0 0 0 bimaculoides]

LOW QUALITY PROTEIN: 5524 annexin A4-like [Crassostrea Protein-Protein 1 0 0 virginica]

PREDICTED: heterogeneous 5634 nuclear ribonucleoprotein D-like-A Metabolism 2 0 0 isoform X1 [Octopus bimaculoides]

PREDICTED: LOW QUALITY PROTEIN: eukaryotic translation 5636 Metabolism 2 0 0 initiation factor 3 subunit K-like [Octopus bimaculoides]

Endothelin-converting enzyme 1 5689 Metabolism 1 0 0 [Crassostrea gigas]

PREDICTED: WW domain-binding 5704 protein 2-like [Octopus Protein-protein 0 3 0 bimaculoides]

PREDICTED: MICOS complex 5751 subunit MIC13-like [Octopus Membrane 0 0 5 bimaculoides]

PREDICTED: microsomal 5834 triglyceride transfer protein large Metabolism 0 0 1 subunit-like [Octopus bimaculoides]

5855 Uncharacterized Uncharacterized 1 0 0

145 PREDICTED: uncharacterized 5857 protein LOC106874393 isoform X7 Uncharacterized 0 0 0 [Octopus bimaculoides]

Glutamine synthetase [Tegillarca 6213 Metabolism 1 0 0 granosa]

PREDICTED: mitochondrial 6327 pyruvate carrier 1-like isoform X1 Ion Transport 0 0 3 [Octopus bimaculoides]

Hypothetical protein 6359 OCBIM_22031876mg [Octopus Uncharacterized 3 0 0 bimaculoides]

Ribonuclease E [Idiomarina 6707 Metabolism 4 0 0 donghaiensis]

PREDICTED: actin-related protein 3 6874 Cytoskeleton 1 0 0 [Lingula anatina]

Sulfotransferase family cytosolic 1B 7189 member 1-like [Octopus Metabolism 1 0 0 bimaculoides]

PREDICTED: importin-4-like 7243 Membrane 1 0 0 [Octopus bimaculoides]

PREDICTED: 26S proteasome non- 7250 ATPase regulatory subunit 2-like Metabolism 1 0 0 [Octopus bimaculoides]

Elongation of very long-chain fatty 7358 Metabolism 0 0 2 acids protein [Sepia officinalis]

PREDICTED: uncharacterized 7560 protein LOC106872137 [Octopus Uncharacterized 0 6 0 bimaculoides]

PREDICTED: CD9 antigen-like 7636 Membrane 3 0 0 [Octopus bimaculoides]

PREDICTED: V-type proton 7661 ATPase subunit C 1-A-like [Octopus Ion Transport 0 1 0 bimaculoides]

146 PREDICTED: alpha-L-fucosidase- 7700 like isoform X1 [Octopus Metabolism 0 1 0 bimaculoides]

PREDICTED: retinol 7926 dehydrogenase 3-like [Octopus Metabolism 1 0 0 bimaculoides]

PREDICTED: calpain-9-like 7930 Metabolism 0 0 1 [Aplysia californica]

PREDICTED: B-cell receptor- 8024 associated protein 31-like [Octopus Membrane 0 4 0 bimaculoides]

PREDICTED: alpha-aminoadipic 8068 semialdehyde dehydrogenase-like Metabolism 1 0 0 [Octopus bimaculoides]

PREDICTED: MICOS complex 8167 subunit Mic60-like [Octopus Membrane 0 0 1 bimaculoides]

Estrogen receptor [Sepiella 8211 Metabolism 0 0 1 maindroni]

PREDICTED: vacuolar protein 8297 sorting-associated protein 13A-like Metabolism 0 0 0 [Octopus bimaculoides]

PREDICTED: dystroglycan-like 8391 Membrane 0 1 0 [Octopus bimaculoides]

PREDICTED: aconitate hydratase, 8480 mitochondrial-like [Octopus Metabolism 0 0 1 bimaculoides]

PREDICTED: ninjurin-2-like 8504 Membrane 5 0 0 isoform X1 [Octopus bimaculoides]

PREDICTED: NADH dehydrogenase [ubiquinone] 1 alpha 8601 subcomplex subunit 9, Metabolism 1 0 0 mitochondrial-like [Octopus bimaculoides]

147 Uncharacterized protein 8635 LOC106879019 [Octopus Uncharacterized 1 0 0 bimaculoides]

PREDICTED: actin-related protein 8652 2/3 complex subunit 5-like [Octopus Cytoskeleton 0 3 0 bimaculoides]

PREDICTED: beta-lactamase 8879 domain-containing protein 2-like Metabolism 0 0 1 [Octopus bimaculoides]

Chain A, Neuronal Complexin 8988 Protein-Protein 4 0 0 SNARE COMPLEX [Loligo Pealei]

PREDICTED: NADH dehydrogenase [ubiquinone] 9022 Metabolism 0 0 2 flavoprotein 2, mitochondrial-like [Octopus bimaculoides]

Hypothetical protein 9059 OCBIM_22029476mg [Octopus Uncharacterized 0 0 0 bimaculoides]

PREDICTED: fatty acid-binding 9223 protein 2, liver-like [Octopus Metabolism 3 0 0 bimaculoides]

PREDICTED: histone H1-delta-like 9293 Nucleosome 4 0 0 [Priapulus caudatus]

PREDICTED: actin-interacting 9333 protein 1-like [Octopus Metabolism 1 0 0 bimaculoides]

PREDICTED: neuronal calcium 9559 sensor 2-like [Octopus Protein-Protein 2 0 0 bimaculoides]

PREDICTED: mitochondrial- 9631 processing peptidase subunit beta- Metabolism 1 0 0 like [Octopus bimaculoides]

PREDICTED: arylacetamide 9661 deacetylase-like isoform X1 Metabolism 0 0 1 [Octopus bimaculoides]

148 PREDICTED: uncharacterized 9662 protein LOC106868256 isoform X2 Uncharacterized 1 0 0 [Octopus bimaculoides]

PREDICTED: trifunctional enzyme 9794 subunit alpha, mitochondrial-like Metabolism 1 0 0 [Octopus bimaculoides]

PREDICTED: acid ceramidase-like 9944 Metabolism 0 1 0 [Octopus bimaculoides]

PREDICTED: F-actin-capping 9948 protein subunit alpha-2-like Cytoskeleton 0 1 0 [Octopus bimaculoides]

PREDICTED: aspartyl/asparaginyl 9967 beta-hydroxylase-like [Biomphalaria Metabolism 3 0 0 glabrata]

PREDICTED: protein-glutamine 10120 gamma-glutamyltransferase K-like Protein-Protein 1 0 0 [Octopus bimaculoides]

PREDICTED: programmed cell 10388 death 6-interacting protein-like Protein-Protein 1 0 0 [Octopus bimaculoides]

PREDICTED: ornithine carbamoyltransferase, 10410 Metabolism 1 0 0 mitochondrial-like isoform X1 [Octopus bimaculoides]

PREDICTED: H(+)/Cl(-) exchange 10478 transporter 3-like isoform X2 Ion Transport 0 1 0 [Octopus bimaculoides]

PREDICTED: eukaryotic translation 10634 initiation factor 3 subunit C-like Metabolism 1 0 0 [Octopus bimaculoides]

PREDICTED: fatty acid-binding 10775 protein, liver-like [Octopus Metabolism 3 0 0 bimaculoides]

149 PREDICTED: peptidyl-prolyl cis- 10871 trans isomerase B-like [Octopus Metabolism 3 0 0 bimaculoides]

Arginase type I-like protein 10874 Metabolism 0 2 0 [Hyriopsis cumingii]

PREDICTED: ribosome-binding 10877 protein 1-like [Octopus Membrane 0 0 0 bimaculoides]

PREDICTED: MAM and LDL- receptor class A domain-containing 11116 Membrane 0 0 0 protein 2-like [Octopus bimaculoides]

Poly endoribonuclease-c-like- 11557 Metabolism 0 0 2 specific [Mytilus galloprovincialis]

Hypothetical protein 11565 OCBIM_22013360mg [Octopus Uncharacterized 2 0 0 bimaculoides]

Thioredoxin reductase 3-like 11686 Metabolism 0 1 0 [Scleropages formosus]

PREDICTED: integrin alpha-6-like 11728 Membrane 0 0 1 [Octopus bimaculoides]

PREDICTED: probable methylmalonate-semialdehyde 11843 dehydrogenase [acylating], Metabolism 0 1 0 mitochondrial [Octopus bimaculoides]

PREDICTED: neurocalcin homolog 12130 Protein-Protein 0 0 2 isoform X2 [Octopus bimaculoides]

PREDICTED: histone deacetylase 12340 11-like isoform X2 [Octopus Metabolism 0 0 2 bimaculoides]

PREDICTED: fatty aldehyde 13086 dehydrogenase-like [Octopus Metabolism 1 0 0 bimaculoides]

150 PREDICTED: carbonyl reductase 13337 [NADPH] 1-like [Octopus Metabolism 0 1 0 bimaculoides]

PREDICTED: cytochrome c oxidase subunit 7A-related protein, 13839 Metabolism 0 3 0 mitochondrial-like [Octopus bimaculoides]

PREDICTED: 40S ribosomal 13901 protein S15a-like [Octopus Metabolism 3 0 0 bimaculoides]

Glucose-methanol-choline 14069 oxidoreductase [Sporothrix Metabolism 3 0 0 insectorum RCEF 264]

PREDICTED: 40S ribosomal 14506 protein S15-like [Octopus Metabolism 0 4 0 bimaculoides]

40S ribosomal protein S3a 14666 Metabolism 0 0 3 [Orbicella faveolata]

Hypothetical protein 14743 OCBIM_22013360mg [Octopus Uncharacterized 3 0 0 bimaculoides]

PREDICTED: cytoplasmic 14937 phosphatidylinositol transfer protein Metabolism 3 0 0 1-like [Octopus bimaculoides]

PREDICTED: general transcription 15124 factor 3C polypeptide 1-like Metabolism 0 0 4 [Octopus bimaculoides]

PREDICTED: maleylacetoacetate 15246 isomerase-like [Octopus Metabolism 2 0 0 bimaculoides]

PREDICTED: iduronate 2-sulfatase- 15589 Metabolism 2 0 0 like [Octopus bimaculoides]

PREDICTED: importin-5-like 16292 Protein-Protein 0 0 0 [Octopus bimaculoides]

151 PREDICTED: thioredoxin-like 16601 Metabolism 4 0 0 [Octopus bimaculoides]

PREDICTED: thioredoxin-related 16981 transmembrane protein 1-like Metabolism 0 4 0 [Octopus bimaculoides]

PREDICTED: gelsolin-like protein 2 17158 Cytoskeleton 1 0 0 [Octopus bimaculoides]

RAD50-interacting protein 1 17371 Protein-Protein 0 0 3 [Labrus bergylta]

PREDICTED: cytochrome P450 17450 Metabolism 3 0 0 20A1-like [Octopus bimaculoides]

PREDICTED: uncharacterized 17477 protein LOC106884065 isoform X1 Uncharacterized 0 1 0 [Octopus bimaculoides]

PREDICTED: histone H1-delta-like 17739 Nucleosome 3 0 0 [Octopus bimaculoides]

Ribosomal protein L12, partial 17988 Metabolism 0 0 3 [Orbicella franksi]

Hypothetical protein 18713 OCBIM_22031876mg [Octopus Uncharacterized 4 0 0 bimaculoides]

PREDICTED: ubiquitin-conjugating 18803 enzyme E2 variant 2-like [Octopus Metabolism 3 0 0 bimaculoides]

PREDICTED: 40S ribosomal 19325 Metabolism 4 0 0 protein S26 [Octopus bimaculoides]

Glutaredoxin [Haliotis diversicolor 20227 Metabolism 4 0 0 supertexta]

PREDICTED: general transcription 20390 factor 3C polypeptide 1-like Metabolism 5 0 0 [Octopus bimaculoides]

PREDICTED: transmembrane 20778 protein 254-like isoform X2 Membrane 0 0 8 [Octopus bimaculoides]

152 PREDICTED: histidine triad 20854 nucleotide-binding protein 3-like Metabolism 5 0 0 [Biomphalaria glabrata]

Table A.2. Proteins from isolated and purified granules (N = 1 MS/MS). Samples were not separated by color; instead they were collected from whole skin sections across the dorsal and ventral regions of four animals, pooled and distributed at random throughout the extraction and identification studies.

Accession # Protein Name Category Peptide Count

11724 Omega-crystallin [Octopus bimaculoides] Crystallin 158

Collagen alpha-4(VI) chain-like [Octopus Extracellular 21216 154 bimaculoides] Matrix

20135 Carboxypeptidase A Metabolism 109 6812 Immunoglobulin E-set Metabolism 103

Voltage-dependent anion-selective channel protein 5905 Ion Transport 98 2-like [Octopus bimaculoides]

11795 Uncharacterized Uncharacterized 98

ADP,ATP carrier protein 3, mitochondrial-like 3200 Membrane 71 [Octopus bimaculoides]

11325 CD63 antigen-like [Aplysia californica] Membrane 52

Glutathione S-transferase Y1-like [Crassostrea 6946 Metabolism 47 gigas]

PREDICTED: hemocyte protein-glutamine 16456 gamma-glutamyltransferase-like isoform X2 Protein-protein 46 [Octopus bimaculoides]

Phosphate carrier protein, mitochondrial-like 5134 Membrane 44 [Octopus bimaculoides]

6813 Vacuolar protein sorting-associated protein 4B Metabolism 37 17802 Tubulin beta-4B chain Cytoskeleton 37

153 Extracellular 5469 Matrilin 2 35 Matrix

6955 Elongation factor 1-alpha Metabolism 35 5143 Translocon-associated protein subunit beta Metabolism 29

PREDICTED: ATP synthase subunit beta, 16139 Metabolism 28 mitochondrial-like [Octopus bimaculoides]

PREDICTED: pyridoxal 5'-phosphate synthase 1684 Metabolism 26 subunit SNZERR-like [Octopus bimaculoides]

9250 Tubulin α-1B Cytoskeleton 24

PREDICTED: casein kinase II subunit alpha 16741 Metabolism 21 isoform X2 [Aplysia californica]

5009 Uncharacterized Prot Metabolism 21 10106 Vitellinogen, open beta-sheet Metabolism 20 10636 Ribonuclease T2 Metabolism 20 13974 Histone H2B Nucleosome 19 13200 Annexin Protein-protein 17

PREDICTED: collagen alpha-1(XII) chain-like Extracellular 8375 16 [Octopus bimaculoides] Matrix

9551 Belongs to the 3-beta-HSD family Metabolism 16

8572 Uncharacterized Uncharacterized 16

8660 Calcium ion binding Metabolism 16 11219 CBN-EMB-9 protein Protein-protein 14 9330 EGF-like domain Membrane 14

PREDICTED: integrin beta-2-like [Octopus 17780 Membrane 14 bimaculoides]

7836 Paramyosin Cytoskeleton 13 20952 ATP synthase subunit alpha Metabolism 13 11343 Ferritin Metabolism 13

154 2093 Elongation factor 1-alpha Metabolism 12

PREDICTED: zonadhesin-like [Octopus 13238 Protein-protein 12 bimaculoides]

6997 Transferrin-like [Octopus bimaculoides] Membrane 12

Extracellular 13241 Collagen alpha-4(VI) chain-like [Lingula anatina] 12 Matrix

9065 MACPF domain containing protein Membrane 12

PREDICTED: prohibitin-like [Octopus 5650 Metabolism 10 bimaculoides]

Collagen alpha-2(IV) chain-like [Octopus Extracellular 9781 10 bimaculoides] Matrix

8515 Isocitrate dehydrogenase [NADP] Metabolism 10

PREDICTED: 60S ribosomal protein L23a-like 1245 Metabolism 9 [Octopus bimaculoides]

8780 Uncharacterized Uncharacterized 8

PREDICTED: laminin subunit alpha-like isoform Extracellular 13237 8 X2 [Lingula anatina] Matrix

8042 Malate dehydrogenase Metabolism 7

PREDICTED: copine-3-like [Octopus 10121 Membrane 7 bimaculoides]

PREDICTED: zinc metalloproteinase nas-15-like 9091 Metabolism 6 [Aplysia californica]

PREDICTED: laminin subunit beta-1-like Extracellular 9620 6 [Octopus bimaculoides] Matrix

Complement component C3-like protein 11739 Protein-protein 6 [Euprymna scolopes]

9821 Prohibitin 2 [Sepiella japonica] Metabolism 6

PREDICTED: protein DD3-3-like [Lingula 11898 Metabolism 6 anatina]

155 Extracellular 13274 Laminin subunit gamma-1 5 Matrix

PREDICTED: cathepsin L1-like [Octopus 6049 Metabolism 5 bimaculoides]

10075 Uncharacterized Prot Membrane 5

Complement component C3-like protein 11735 Protein-protein 5 [Euprymna scolopes]

9086 Spectrin beta chain-like [Octopus bimaculoides] Cytoskeleton 5

Basement membrane-specific heparan sulfate Extracellular 13189 4 proteoglycan core protein Matrix

PREDICTED: cysteine-rich secretory protein 3- 7854 Protein-protein 4 like [Octopus bimaculoides]

6681 EGF-like domain Membrane 4

PREDICTED: protein disulfide-isomerase 2-like 8129 Metabolism 4 [Octopus bimaculoides]

PREDICTED: inter-alpha-trypsin inhibitor heavy 11240 Metabolism 3 chain H5-like [Octopus bimaculoides]

10357 Apolipoprotein B Protein-protein 3

PREDICTED: neurotrypsin-like [Limulus 10585 Metabolism 3 polyphemus]

Basement membrane-specific heparan sulfate Extracellular 12393 3 proteoglycan core protein Matrix

PREDICTED: beta-mannosidase-like [Octopus 12352 Metabolism 3 bimaculoides]

P85084 Endochitinase Contaminant 233

P00784 Papain Contaminant 134 P35059 Histone H4 Nucleosome 127 P05994 Papaya proteinase 4 Contaminant 60 P09870 Clostripain Contaminant 19

156 Table A.3. Proteins in extracted granules; (N = 1 MS/MS). Samples were not separated by color; instead they were collected from whole skin sections across the dorsal and ventral regions of four animals, pooled and distributed at random throughout the extraction and identification studies.

Accession # Protein Name Category Peptide Count 11724 Omega-crystallin [Octopus bimaculoides] Crystallin 32

Voltage-dependent anion-selective channel 5905 Ion Transport 25 protein 2-like [Octopus bimaculoides]

Collagen alpha-4(VI) chain-like [Octopus Extracellular 21216 18 bimaculoides] Matrix

Glutathione S-transferase Y1-like [Crassostrea 6946 Metabolism 8 gigas]

6955 Elongation factor 1-alpha Metabolism 7

Extracellular 5469 Matrilin 2 6 Matrix

6812 Immunoglobulin E-set Metabolism 5

PREDICTED: inter-alpha-trypsin inhibitor 11240 Metabolism 4 heavy chain H5-like [Octopus bimaculoides]

PREDICTED: collagen alpha-4(VI) chain-like Extracellular 6680 2 [Octopus bimaculoides] Matrix

9330 EGF-like domain Membrane 2

Basement membrane-specific heparan sulfate Extracellular 13189 2 proteoglycan core protein Matrix

P85084 Endochitinase Contaminant 70 P00784 Papain Contaminant 38

P05994 Papaya proteinase 4 Contaminant 27 C3K2Y6 50S ribosomal protein L1 Contaminant 20 C3K2X8 Elongation factor Tu Contaminant 11 P00761 Trypsin Contaminant 30 Q3K603 50S ribosomal protein L6 Contaminant 22

157 Q3K5Y8 50S ribosomal protein L3 Contaminant 17

Succinyl-CoA ligase [ADP-forming] subunit Q3KFU6 Contaminant 8 beta

Q4K556 30S ribosomal protein S4 Contaminant 11

Table A.4. Proteins Found in Pigment; (N = 1 MS/MS). Samples were not separated by color; instead they were collected from whole skin sections across the dorsal and ventral regions of four animals, pooled and distributed at random throughout the extraction and identification studies.

Accession # Protein Name Category Peptide Count Voltage-dependent anion-selective channel 5905 Ion Transport 31 protein 2-like [Octopus bimaculoides] 11724 Omega-crystallin [Octopus bimaculoides] Crystallin 22 Collagen alpha-4(VI) chain-like [Octopus Extracellular 21216 16 bimaculoides] Matrix Extracellular 5469 Matrilin 2 8 Matrix PREDICTED: Inter-alpha-trypsin inhibitor 11240 Metabolism 7 heavy chain H5-like [Octopus bimaculoides] 9330 EGF-like domain Membrane 2 10106 Vitellogenin Metabolism 1 P35059 Histone H4 Nucleosome 103 P85084 Endochitinase Contaminant 79 P00784 Papain Contaminant 35 P05994 Papaya proteinase 4 Contaminant 19 P14080 Chymopapain Contaminant 12 P02769 Serum albumin Contaminant 4

158 A2.1.Coordinates and energies of the closed-shell substitution reaction, (6a→7)-S0 6a – Reactant 0 1 C 0.77275500 4.78025300 1.13758300 C -0.42216400 4.13327800 0.80332300 C 1.98879800 4.12022800 1.02116500 C -0.41595000 2.81954200 0.34638400 C 0.80639900 2.11211900 0.26764400 C 1.98540600 2.80225100 0.58448800 O 3.18384200 2.15790000 0.46086400 C 3.23067300 0.86620600 0.07315000 C 1.96377700 0.13323300 -0.06744000 N 0.82381000 0.77071000 -0.02603000 C 4.44401500 0.31663600 -0.18261500 C 4.53807800 -1.04870200 -0.59764000 C 3.25398800 -1.85484600 -0.65402400 C 2.02126100 -1.29127300 -0.30121900 C -1.71442100 2.23455900 -0.10830800 O -2.71488800 2.32776100 0.58982900 C -1.76194600 1.69159700 -1.52088400 C -3.07856600 1.06104800 -1.97083000 N -4.28283200 1.88317500 -1.60248600 C -3.33615600 -0.37998200 -1.44323800 O -2.34921900 -1.15243000 -1.53316600 O -4.48887400 -0.62769200 -1.03935000 C 0.86280300 -2.17144100 -0.09943400 O 0.85486100 -3.32289600 -0.55852600 C -0.28896900 -1.72430800 0.77985500 C -1.22553300 -2.86311700 1.14969300 N -1.94573600 -3.37159500 -0.06211500 C -2.29268000 -2.48171900 2.22086300 O -3.44359900 -2.94768500 2.01963500 O -1.88613200 -1.79647600 3.18146500 O 5.59597200 -1.61110500 -0.89048800 N 3.44443700 -3.11555500 -1.01166300 H 0.75069400 5.80723400 1.47844800 H -1.36306800 4.66368900 0.87277400 H 2.92749100 4.60225000 1.26210100 H 5.34958600 0.90200900 -0.09528600 H -1.48704300 2.52634000 -2.17625800 H -0.97341600 0.94679200 -1.63613700 H -3.06926800 0.98614000 -3.05861300

159 H -4.12293300 2.29913300 -0.67363100 H -4.51015800 2.60968300 -2.28007600 H -5.05626800 1.20599500 -1.50446500 H -0.83480900 -0.93416000 0.26738800 H 0.10004400 -1.27805200 1.69587700 H -0.65763300 -3.70426500 1.55220300 H -2.80869500 -3.81375100 0.27529600 H -2.21490200 -2.56473600 -0.69258100 H -1.35796900 -4.01897500 -0.58587800 H 4.39614600 -3.41520800 -1.18128700 H 2.64932700 -3.73967600 -1.07703600

SCF energy: -1594.422432 Hartree zero-point correction: + 0.377883 Hartree enthalpy correction: + 0.406703 Hartree free energy correction: + 0.318422 Hartree

First five vibrational frequencies (cm-1): 17.57, 31.43, 38.80, 47.22, 48.44

TS (6a→7) – Transition State 0 1 C -3.18655100 3.98769400 0.81380200 C -3.67653600 2.69515700 0.57372500 C -1.83515500 4.28021400 0.68537300 C -2.80742600 1.67271400 0.22924500 C -1.42855200 1.94845700 0.08459700 C -0.97196300 3.25682600 0.31553400 O 0.35305200 3.53976100 0.16573400 C 1.22535800 2.56776200 -0.19510700 C 0.68540500 1.23725100 -0.47963000 N -0.57483400 0.95902000 -0.32927200 C 2.54793300 2.86585600 -0.25966000 C 3.50145100 1.85166800 -0.61919400 C 2.94776500 0.50960300 -0.94877900 C 1.63002100 0.20596200 -0.86322800 C -3.31759800 0.28954900 -0.04828100 O -4.17723600 0.11157700 -0.89468200 C -2.75358600 -0.84776800 0.76989800 C -2.97129000 -2.22888200 0.16607800 N -2.57041200 -2.27950000 -1.28137800 C -4.42175300 -2.80107100 0.27109200

160 O -4.77469100 -3.51550000 -0.70069500 O -5.03822800 -2.53617700 1.32040200 C 1.20369700 -1.20390200 -1.05732500 O 0.17027500 -1.49541200 -1.63066300 C 2.04856600 -2.33412000 -0.45917700 C 3.46006900 -2.08091700 0.00811900 N 3.50345600 -3.91378000 1.27974100 C 3.75156600 -1.26178800 1.28771200 O 4.95769800 -1.00940100 1.49644400 O 2.74320200 -0.94020000 1.95358900 O 4.72454000 2.02032800 -0.66374700 N 3.87237400 -0.49554100 -1.25493800 H -3.87420400 4.77322900 1.09976900 H -4.73487000 2.49050200 0.67056400 H -1.44709300 5.27560100 0.85706200 H -3.28419400 -0.82769600 1.72860800 H -1.70264700 -0.66232900 0.98280100 H -2.32163900 -2.93770300 0.68241500 H -3.02603300 -1.51677400 -1.78886200 H -2.97032900 -3.15031300 -1.65398900 H -1.55439100 -2.21829900 -1.43267700 H 2.02130600 -3.15579600 -1.17593300 H 1.45768800 -2.63243200 0.41081400 H 4.27594300 -2.59073300 -0.47400000 H 4.44959000 -4.01826300 1.63431700 H 2.87271600 -3.80440400 2.06853500 H 3.25228300 -4.75606700 0.76979500 H 4.82866200 -0.17839000 -1.09359300 H 3.78113200 -0.90234200 -2.18138900 H 2.90457900 3.85942900 -0.02312000

SCF energy: -1594.330401 Hartree zero-point correction: + 0.373301 Hartree enthalpy correction: + 0.403395 Hartree free energy correction: + 0.311629 Hartree

First five vibrational frequencies (cm-1): -466.15, 19.57, 22.21, 33.53, 43.06

7 – Product

161 -1 1 C -4.27770000 -3.26990100 -0.05172000 C -4.43366400 -1.89920900 -0.28739100 C -3.01459000 -3.81199000 0.14985100 C -3.33163700 -1.05248400 -0.29802600 C -2.03429000 -1.58274900 -0.10970100 C -1.91788400 -2.96166600 0.11155900 O -0.67392700 -3.50014700 0.28369100 C 0.42623500 -2.72171700 0.23812100 C 0.26183500 -1.28026000 -0.03100500 N -0.92975000 -0.76880500 -0.19739900 C 1.62558100 -3.32978300 0.43566100 C 2.83507400 -2.57112000 0.41540500 C 2.69686600 -1.08590900 0.17755500 C 1.45885800 -0.47713100 -0.10487500 C -3.53229400 0.41226100 -0.52979900 O -4.17346900 0.80516100 -1.48959000 C -2.97840400 1.37025100 0.50479400 C -2.89871300 2.80023600 0.00826000 N -1.91709100 2.91371900 -1.12391200 C -2.48237200 3.83831200 1.09636300 O -1.65224100 4.70309600 0.71355000 O -3.04216600 3.72351100 2.20302000 C 1.47918900 0.90597300 -0.57103100 O 0.48629300 1.58369100 -0.84411000 C 2.85780100 1.50337800 -0.83032300 C 3.89618500 1.03024500 0.17452300 C 5.35832000 1.47036200 -0.13229700 O 6.23708900 0.58817300 0.02965000 O 5.50556400 2.66373500 -0.47490300 O 3.96087100 -3.04585300 0.58715500 N 3.82698100 -0.41722700 0.26440000 H -5.14698800 -3.91440700 -0.02821000 H -5.42121200 -1.48520800 -0.44649100 H -2.86679000 -4.86991000 0.32506200 H 1.67731300 -4.39482000 0.61971600 H -2.00841400 1.01530100 0.85254300 H -3.65432500 1.33767100 1.36528400 H -3.86561500 3.11398700 -0.38674200 H -1.11059200 2.26910000 -1.01224400 H -2.34862500 2.75488400 -2.03315400 H -1.54396900 3.87457800 -1.04885100 H 3.15303900 1.20056000 -1.84338900

162 H 2.77681700 2.58726200 -0.81765100 H 3.64907700 1.43994800 1.16516200 H 4.69880500 -0.91625800 0.42411300

SCF energy: -1537.364127 Hartree zero-point correction: + 0.324380 Hartree enthalpy correction: + 0.351134 Hartree free energy correction: + 0.265626 Hartree

First five vibrational frequencies (cm-1): 14.16, 19.34, 33.85, 41.96, 48.95

A2. 2. Coordinates and energies of the open-shell substitution reaction, (6a→7)-D1 6a – Reactant 1 2 C 4.66252900 2.43715600 0.05364900 C 4.45699300 1.05097500 -0.09622600 C 3.59918700 3.33046100 0.09381800 C 3.18192300 0.54856700 -0.24475800 C 2.06662900 1.43838700 -0.19413500 C 2.31465700 2.82637800 -0.01688100 O 1.27261000 3.69473200 0.06665000 C -0.00387700 3.24455700 0.02569300 C -0.20250100 1.80099500 -0.14541300 N 0.81340200 0.95256000 -0.25155400 C -1.02295300 4.12693000 0.14840500 C -2.38364600 3.65903500 0.14991000 C -2.60290200 2.15197100 0.02385700 C -1.50307200 1.28339900 -0.16550400 C 2.93854100 -0.91327400 -0.47766900 O 2.30030300 -1.29204900 -1.44279400 C 3.53032000 -1.89087900 0.51728200 C 2.77605800 -3.21477900 0.56585800 N 2.82548800 -3.95731000 -0.74182800 C 1.27228100 -3.05987500 0.94345300 O 0.98030300 -2.15656300 1.73699700 O 0.50655100 -3.90955800 0.39699100 C -1.69549000 -0.19697600 -0.38062000 O -1.67830600 -0.64318600 -1.51045800 C -1.88957500 -1.03756200 0.84481000 C -2.72157000 -2.28105300 0.57627200 N -2.00054800 -3.23154100 -0.33032400

163 C -4.12080900 -1.99089500 -0.04773000 O -4.57695800 -0.82070600 0.09141700 O -4.66534500 -2.95761400 -0.60563700 O -3.37288200 4.37192800 0.26169500 N -3.83608700 1.73514500 0.10992200 H 5.67288100 2.81424200 0.14455500 H 5.31393800 0.39074300 -0.11685600 H 3.75174900 4.39352400 0.22290900 H -0.83684500 5.18573900 0.26460400 H 4.58547500 -2.04562500 0.26232000 H 3.51407900 -1.45324100 1.51450000 H 3.23480200 -3.86379200 1.31222800 H 2.66765900 -3.29104900 -1.50760600 H 3.69775700 -4.46328900 -0.89491800 H 2.02398600 -4.60489500 -0.73002200 H -2.35801200 -0.45198500 1.63609700 H -0.88576500 -1.30527200 1.21371100 H -2.88376200 -2.81225100 1.51533500 H -1.07512100 -3.56143800 0.06603100 H -2.62222100 -4.01678200 -0.53548000 H -1.80244100 -2.75188600 -1.21363600 H -4.11500000 0.71473200 0.09156600 H -4.54549500 2.45357800 0.24784000

SCF energy: -1594.205278 Hartree zero-point correction: + 0.377844 Hartree enthalpy correction: + 0.406253 Hartree free energy correction: + 0.319924 Hartree

First five vibrational frequencies (cm-1): 26.25, 47.53, 50.96, 58.22, 66.70

164 TS (6a→7) – Transition State 1 2 C -3.43501600 3.62309700 0.59198900 C -3.80199000 2.34214200 0.15806000 C -2.10102100 3.99394100 0.67390600 C -2.84046500 1.40236000 -0.18621500 C -1.47064500 1.76458700 -0.12225000 C -1.13640000 3.06206400 0.31289200 O 0.17132600 3.43379900 0.38686300 C 1.15236800 2.56924700 0.04127600 C 0.74649300 1.26150500 -0.46850400 N -0.49833400 0.89040300 -0.52334000 C 2.44833700 2.94756000 0.19633100 C 3.50813200 2.04737300 -0.16087200 C 3.09185200 0.74317300 -0.74223600 C 1.80145900 0.35227300 -0.86718800 C -3.30740500 0.04152700 -0.59753600 O -4.39473600 -0.07280000 -1.14919000 C -2.47548700 -1.18782800 -0.29401900 C -3.32997100 -2.42145200 -0.03353400 N -4.12930000 -2.83918300 -1.23781100 C -4.35347100 -2.26697100 1.14127100 O -3.98843300 -1.55828700 2.09638000 O -5.42249700 -2.91140800 0.98775700 C 1.51350900 -1.02276200 -1.35749100 O 0.62805600 -1.27931000 -2.13789100 C 2.33454100 -2.19252400 -0.77129300 C 3.73552400 -1.99119100 -0.24510400 N 3.80489400 -3.95849300 0.74502100 C 3.96586500 -1.43474000 1.11696700 O 5.09393500 -1.35775000 1.66631800 O 3.04553500 -0.96065800 1.82416400 O 4.71563400 2.27192100 -0.02179800 N 4.12663300 -0.14071100 -1.09459300 H -4.20313800 4.33336600 0.86938900 H -4.84622800 2.06605100 0.10347500 H -1.79809300 4.97876600 1.00431800 H -1.85130800 -1.01722700 0.57938800 H -1.78763800 -1.34676400 -1.13006400 H -2.67824400 -3.25996500 0.21442200 H -4.49314800 -1.99469700 -1.69573000 H -3.61192000 -3.40847300 -1.90613700 H 1.69838800 -2.55187900 0.04270500

165 H 2.35411400 -2.96477700 -1.53813100 H 4.58965500 -2.37497900 -0.77581000 H 4.14888800 -0.39864400 -2.07885300 H 5.03454300 0.24883400 -0.82420500 H 2.69784000 3.91972300 0.59969100 H 3.59272400 -4.63589000 0.01650000 H 4.74594800 -4.13752400 1.08687900 H 3.14906300 -4.09097400 1.51170800 H -4.93650000 -3.35339800 -0.84401800

SCF energy: -1594.086751 Hartree zero-point correction: + 0.373340 Hartree enthalpy correction: + 0.403204 Hartree free energy correction: + 0.310684 Hartree

First five vibrational frequencies (cm-1): -474.72, 17.93, 21.80, 29.55, 31.86

A2.3. Coordinates and energies of the closed-shell addition reaction, (6b→8)-S0 6b – Reactant -1 1 C -0.89001400 -4.46328200 1.19103500 C -1.72475200 -3.34848800 1.06089000 C 0.46603200 -4.35310300 0.92406300 C -1.22398500 -2.12004900 0.64314000 C 0.16219700 -1.97352300 0.38672600 C 0.96276000 -3.11555500 0.53606100 O 2.30073400 -3.01742600 0.29908900 C 2.86362200 -1.83697200 -0.02215600 C 2.01169300 -0.63198500 -0.07114700 N 0.71516200 -0.74869600 0.08463500 C 4.19218100 -1.85188400 -0.29661900 C 4.86386700 -0.64166400 -0.62263200 C 4.04570500 0.63406800 -0.59280000 C 2.66677300 0.64511200 -0.30330100 C -2.21981600 -1.02033600 0.45527700 O -3.04554600 -0.79191200 1.32818400 C -2.23558000 -0.27683500 -0.85875800 C -3.59553200 0.31585500 -1.20581700 N -3.99044000 1.37087600 -0.22112400 C -4.75117000 -0.72466800 -1.32624100

166 O -5.89294900 -0.28537200 -1.04068800 O -4.42919400 -1.86285700 -1.72832900 C 1.98772800 1.95428800 -0.21140600 O 2.61806800 2.99852500 -0.43667600 C 0.52528900 2.07426900 0.14535200 C 0.09597600 3.50481100 0.47196100 N 0.72956800 3.95883700 1.71497100 C -1.44395100 3.56992100 0.56467900 O -1.97412000 4.04077800 1.58935300 O -2.05989000 3.13211600 -0.46293100 O 6.06378500 -0.56604900 -0.90572300 N 4.78388300 1.70149200 -0.85225700 H -1.30378000 -5.41463500 1.49966200 H -2.78385900 -3.43855300 1.26381600 H 1.14196400 -5.19323700 1.01817500 H -1.92522900 -0.94980800 -1.65680800 H -1.48054100 0.50891700 -0.80237100 H -3.51539500 0.81650800 -2.17319400 H -4.96566600 1.61688200 -0.40333400 H -3.94859900 0.95842300 0.71524400 H -3.32606200 2.20924500 -0.27094300 H 0.27946000 1.40594500 0.97037600 H -0.03690600 1.71399300 -0.71828900 H 0.36369100 4.14471600 -0.37883500 H 0.13945600 4.66679800 2.14040200 H 1.63244600 4.37104600 1.50762800 H 5.76807500 1.54724400 -1.03412000 H 4.34231500 2.61495200 -0.83984900 H 4.75016600 -2.77830800 -0.26922300

SCF energy: -1593.956732 Hartree zero-point correction: + 0.362643 Hartree enthalpy correction: + 0.391550 Hartree free energy correction: + 0.302306 Hartree

First five vibrational frequencies (cm-1): 15.79, 24.73, 35.58, 43.33, 58.38

167 TS (6b→8) – Transition State -1 1 C 2.94251800 3.95471400 0.50409800 C 3.47297500 2.73491400 0.10394800 C 1.56085600 4.11237400 0.58916400 C 2.66633300 1.64043000 -0.24154800 C 1.24851400 1.79103900 -0.16733100 C 0.75603900 3.03810700 0.26301900 O -0.60217600 3.20517800 0.41384100 C -1.46358400 2.22105300 0.11863400 C -0.93199500 0.96943700 -0.45635500 N 0.38453000 0.77713200 -0.47927100 C -2.77271300 2.42558600 0.43020600 C -3.75890300 1.44979900 0.10254200 C -3.28250100 0.22830400 -0.68232800 C -1.85314900 -0.01072300 -0.86985800 C 3.40147400 0.41203700 -0.69632200 O 4.62129900 0.37549900 -0.58233700 C 2.70932200 -0.78064300 -1.34266800 C 2.42094400 -1.94712500 -0.37181600 N 1.45034400 -1.52347400 0.68268200 C 3.70218600 -2.48483000 0.32890600 O 3.82090800 -2.18779500 1.54647200 O 4.47875400 -3.14823600 -0.38942100 C -1.45293000 -1.33387700 -1.33760400 O -0.38781100 -1.58377100 -1.90393400 C -2.39207100 -2.47702000 -0.95421200 C -2.86736000 -2.32927000 0.50343800 N -3.69984800 -1.12643200 0.69002700 C -1.66666900 -2.25874600 1.49177400 O -1.73054800 -1.40597500 2.40195500 O -0.73277300 -3.07982100 1.27384200 O -4.95944700 1.54756300 0.36000000 N -4.13102400 -0.01991900 -1.71609700 H 3.59719800 4.77988700 0.75279300 H 4.54449500 2.60629300 0.03804900 H 1.10643000 5.04046400 0.91294300 H 3.40591900 -1.16617300 -2.08772700 H 1.77533800 -0.49600700 -1.81996500 H 1.96145800 -2.74760200 -0.94695900 H 2.01367600 -1.32851000 1.52156600 H 0.93182300 -0.68312300 0.34827200 H 0.74061000 -2.25118800 0.92696900

168 H -1.82845500 -3.40160300 -1.05078500 H -3.25324100 -2.55085200 -1.62193700 H -3.44967900 -3.21504000 0.77173800 H -4.69663700 -1.30152000 0.60233000 H -3.48104100 -0.75172100 1.61710800 H -3.90162500 -0.70678400 -2.41586000 H -5.11343200 0.16914100 -1.56923500 H -3.08222700 3.33869400 0.92183900

SCF energy: -1593.930211 Hartree zero-point correction: + 0.364200 Hartree enthalpy correction: + 0.391383 Hartree free energy correction: + 0.308356 Hartree

First five vibrational frequencies (cm-1): -301.01, 23.10, 29.57, 42.65, 66.45 8 – Product -1 1 C 3.26789100 3.99924300 0.59340800 C 3.73994100 2.68847500 0.53332600 C 1.90737000 4.25004900 0.44104800 C 2.87466000 1.62394700 0.28068700 C 1.48306200 1.84887800 0.12799000 C 1.05417400 3.18022300 0.22583000 O -0.28887900 3.44670600 0.13563200 C -1.17881500 2.45448500 -0.03194700 C -0.68914900 1.06289700 -0.18761200 N 0.60387700 0.80631400 -0.05819300 C -2.48849300 2.82374300 -0.00149100 C -3.51665600 1.86191300 -0.18467700 C -3.10362300 0.46555500 -0.69012200 C -1.66575400 0.06120600 -0.45787200 C 3.44661200 0.25151800 0.21518500 O 4.18652400 -0.15668000 1.09921900 C 3.13404000 -0.60508800 -0.99346000 C 3.03200200 -2.08973200 -0.66202000 N 2.10953600 -2.28837700 0.50110100 C 4.37289700 -2.83453800 -0.39470300 O 5.31500600 -2.54888100 -1.16308300 O 4.33771200 -3.68985200 0.52468300 C -1.38372000 -1.32102400 -0.64633400 O -4.72407900 2.08479000 -0.04698400

169 N -4.01706700 -0.48270700 0.06916500 C -2.56561000 -2.21942400 -0.99711500 O -0.26510400 -1.87937400 -0.56344200 C -3.79642200 -1.95626900 -0.12866200 C -3.63138700 -2.56081100 1.30512200 O -3.59232000 -1.71579300 2.24212900 O -3.54877900 -3.79936900 1.36913900 N -3.48166600 0.36566500 -2.10072600 H 3.95321900 4.81902500 0.76565800 H 4.79534400 2.48354100 0.66227000 H 1.49955100 5.25137300 0.50204600 H -2.75683400 3.85403200 0.19453300 H 2.22800400 -0.25035700 -1.47816800 H 3.97119000 -0.47752800 -1.68743800 H 2.55714000 -2.59756400 -1.50408600 H 2.15961300 -3.27099600 0.77844100 H 2.43339600 -1.75017200 1.30523700 H 1.13191700 -2.00304000 0.23656800 H -4.98108400 -0.20258500 -0.11931400 H -3.86255200 -0.39561200 1.10319800 H -2.81767500 -2.07344700 -2.04784500 H -2.26122100 -3.25376000 -0.85273600 H -4.68699800 -2.37813500 -0.58922100 H -2.70378600 0.64006900 -2.68901800 H -4.28075300 0.95503400 -2.31737600

SCF energy: -1593.932610 Hartree zero-point correction: + 0.366081 Hartree enthalpy correction: + 0.393997 Hartree free energy correction: + 0.306814 Hartree

First five vibrational frequencies (cm-1): 10.57, 20.18, 29.78, 50.50, 51.32

170 A2.4. Coordinates and energies of the open-shell addition reaction, (6b→8)-D1 6b – Reactant 0 2 C -2.20743800 4.73461200 0.00436800 C -2.85513600 3.48522200 -0.07588100 C -0.81971300 4.84404400 0.05219000 C -2.11474600 2.32368000 -0.07231300 C -0.69450000 2.40557900 -0.01777800 C -0.07204300 3.68109400 0.02883100 O 1.28636000 3.76687100 0.04332800 C 2.04770100 2.64573100 -0.00034700 C 1.34993900 1.35663000 -0.04834600 N 0.02597500 1.27238000 -0.05089900 C 3.39759000 2.75334000 0.00654900 C 4.21959600 1.56969100 -0.01942900 C 3.50288900 0.21993500 -0.04453600 C 2.08962800 0.16768200 -0.06270900 C -2.75823300 0.96363800 -0.21429200 O -3.37779600 0.70277400 -1.22484600 C -2.62205100 -0.00394900 0.93929100 C -3.00425800 -1.44563500 0.62130600 N -2.16523800 -2.06985700 -0.45785400 C -4.50543600 -1.64037200 0.22271800 O -4.70291100 -2.39216300 -0.76634700 O -5.33644500 -1.07022600 0.95422500 C 1.34355900 -1.13636800 -0.08639400 O 0.70246000 -1.43853200 -1.07024900 C 1.43956100 -2.00092300 1.14724300 C 1.84954500 -3.43206100 0.77440100 N 3.16095600 -3.40723400 0.08309900 C 0.74627700 -4.12981700 -0.07527800 O -0.43524800 -4.01208300 0.37013500 O 1.11275500 -4.75840100 -1.08554200 O 5.44403400 1.56676600 -0.01753600 N 4.24962000 -0.84980200 -0.06194900 H -2.80516300 5.63698100 0.01953100 H -3.93489100 3.44319600 -0.13209100 H -0.32667100 5.80579800 0.09571500 H -3.30763100 0.35330400 1.71590000 H -1.62011000 0.06778300 1.36213500 H -2.84413600 -2.03861300 1.52251400 H -1.61592100 -1.39781500 -0.99258400

171 H -1.50210800 -2.81216200 -0.11262800 H -2.87548100 -2.50129000 -1.08214600 H 0.45236800 -2.01489000 1.61598400 H 2.14046800 -1.57041000 1.86396800 H 1.92616600 -4.00211200 1.70336300 H 3.84295500 -3.94991500 0.60452200 H 3.02959200 -3.89513200 -0.80472300 H 5.25671400 -0.69290000 -0.04890300 H 3.86008100 -1.86015100 -0.05972100 H 3.88252200 3.71935400 0.03947900

SCF energy: -1593.740494 Hartree zero-point correction: + 0.362057 Hartree enthalpy correction: + 0.390601 Hartree free energy correction: + 0.303037 Hartree

First five vibrational frequencies (cm-1): 23.74, 38.40, 43.82, 53.01, 55.61 TS (6b-8) – Transition 0 2 C 1.51834900 4.61325000 0.44013500 C 2.37498200 3.60478000 -0.03794500 C 0.15277600 4.39061700 0.60027300 C 1.86688900 2.36279500 -0.35813100 C 0.47496600 2.11305700 -0.23036400 C -0.35158600 3.14694600 0.26759400 O -1.68576300 2.91210000 0.43337400 C -2.21554500 1.71130900 0.11299100 C -1.33393400 0.70768200 -0.50877300 N -0.01770700 0.91772400 -0.61843700 C -3.52371000 1.50311900 0.40216700 C -4.16529100 0.27094200 0.05299200 C -3.29930700 -0.79655800 -0.63047400 C -1.88467800 -0.50708600 -0.91414700 C -1.01211800 -1.64826900 -1.31547300 O -0.15156300 -1.56031100 -2.16954600 C -1.24493300 -2.90457400 -0.49479100 C -1.65619100 -2.55854400 0.94052000 N -2.97728000 -1.89742900 0.97399400 C -0.63942000 -1.61805100 1.66332100 O 0.57752800 -1.80313000 1.40017700 O -1.13200300 -0.78306800 2.44601900

172 O -5.34927100 0.01767600 0.23577600 N -4.00080600 -1.51862800 -1.51526500 H 1.92924600 5.58145500 0.69597500 H 3.43425200 3.80257700 -0.14017900 H -0.50930900 5.15985500 0.97499700 H -2.01406900 -3.51960600 -0.97362000 H -0.33060000 -3.49386500 -0.49845500 H -1.70859500 -3.48039700 1.52599900 H -3.76165700 -2.54593500 0.96491200 H -2.99457400 -1.31119000 1.81486400 H -5.00276600 -1.58064300 -1.38545100 H -3.57089300 -2.14980000 -2.17275900 C 2.74038500 1.23241200 -0.83582700 O 3.20079100 1.23382500 -1.95859600 C 3.05141800 0.16843500 0.19255700 H 2.21109900 0.04033700 0.87775600 H 3.88057100 0.57041600 0.78841300 C 3.50523300 -1.16049700 -0.38734600 H 4.00592300 -0.99070900 -1.34237800 N 2.34504800 -2.07134200 -0.65346200 H 2.74292800 -3.02178700 -0.61560300 H 1.62698600 -2.00640800 0.10874500 C 4.51452500 -1.94723100 0.51243300 O 5.38751000 -1.26554800 1.08274900 O 4.35502600 -3.19474700 0.51803100 H 1.87260700 -1.90466400 -1.54401200 H -4.11084200 2.27649800 0.87884100

SCF energy: -1593.735982 Hartree zero-point correction: + 0.363294 Hartree enthalpy correction: + 0.391124 Hartree free energy correction: + 0.305940 Hartree

First five vibrational frequencies (cm-1): -238.71, 28.17, 42.77, 46.82, 53.68

8 – Product 0 2 C 2.52302000 4.22774900 0.76514100 C 3.15856300 2.97287600 0.73470800 C 1.15930400 4.35767600 0.51447900 C 2.43473400 1.83904200 0.42742300

173 C 1.03848300 1.93875800 0.17424900 C 0.43360800 3.21521900 0.22959400 O -0.91039200 3.32176100 0.02638900 C -1.66062000 2.22387700 -0.22448600 C -0.97977900 0.91814200 -0.31154600 N 0.33117100 0.81725100 -0.07310200 C -2.99641400 2.40030300 -0.36384400 C -3.85822400 1.27823400 -0.58368700 C -3.22023500 -0.11212000 -0.83926400 C -1.73209000 -0.21732300 -0.61687100 C 3.07287300 0.47898400 0.37271500 O 3.55603800 -0.01509400 1.37493700 C 3.08793700 -0.21488700 -0.96735400 C 3.45546400 -1.68934700 -0.90971100 N 2.56771000 -2.43654300 0.04492700 C 4.93757400 -2.01756300 -0.54491600 O 5.79606500 -1.24011400 -1.00440900 O 5.09253300 -3.06870200 0.12531900 C -1.16519400 -1.57447000 -0.74903700 O -5.08299900 1.33746700 -0.62160000 N -3.91534500 -1.04162100 0.14558300 C -2.15904200 -2.70489400 -0.59682500 O 0.01535500 -1.79441500 -0.98632700 C -3.27284000 -2.38543400 0.39455600 C -2.75515600 -2.32202700 1.87538200 O -2.13330900 -3.31342400 2.28093500 O -3.04467700 -1.25327000 2.48337000 N -3.55219700 -0.56537100 -2.17878800 H 3.10475300 5.11165300 0.99288400 H 4.21946200 2.90116100 0.93636600 H 0.66312600 5.31839300 0.55103800 H -3.43475000 3.38458100 -0.26412100 H 2.12448000 -0.06614900 -1.45517200 H 3.84103900 0.29476300 -1.57738800 H 3.27316500 -2.12817500 -1.89230700 H 2.99657600 -3.35870700 0.18401100 H 2.57830000 -1.97425300 0.95730000 H 1.59571900 -2.49664700 -0.28971900 H -4.89507000 -1.11715900 -0.13057100 H -3.85666900 -0.64259800 1.12621700 H -2.57160900 -2.91814200 -1.58580500 H -1.62741700 -3.58820600 -0.24751500 H -4.05501900 -3.13938700 0.33497600

174 H -2.84685100 -0.27968400 -2.84989200 H -4.44931100 -0.18790900 -2.47379200

SCF energy: -1593.736579 Hartree zero-point correction: + 0.365160 Hartree enthalpy correction: + 0.393574 Hartree free energy correction: + 0.305045 Hartree

First five vibrational frequencies (cm-1): 18.07, 18.75, 31.38, 48.44, 58.77

A2.5. Coordinates and energies of the final product, H2Xa

H2Xa, Singlet -1 1 C 3.29769200 3.64251900 -0.38076100 C 3.67551200 2.32474100 -0.49117000 C 1.95275000 3.95667900 -0.12331700 C 2.73826600 1.26805900 -0.36441500 C 1.02596500 2.95142400 0.01539800 C 1.37194700 1.58518500 -0.11589400 C 3.17019300 -0.12042000 -0.49863900 N 0.37790400 0.65144900 0.00645200 O -0.26875800 3.32678800 0.33127300 C -1.26778800 2.37734700 0.23197800 C -0.96318400 1.03141700 0.08690000 C -2.57871300 2.84067300 0.31415400 C -2.02473700 0.09379400 0.02359900 C -3.62632000 1.94140300 0.26174400 C -3.35529600 0.56857300 0.11736400 O -4.94584000 2.28992800 0.34063300 N -4.40290300 -0.31629600 0.06396800 C -1.79621400 -1.35378400 -0.13013200 O 2.36928100 -1.06369000 -0.41551000 C 4.63173100 -0.43633900 -0.78681700 C 5.03118900 -1.86240500 -0.42819100 N 4.21640400 -2.89555800 -1.15597600 C 4.92632800 -2.20727500 1.09518900 O 4.60049200 -3.39741300 1.33993500 O 5.21366400 -1.29108800 1.88689300 O -0.64044800 -1.83252100 -0.21700400 C -2.97267900 -2.17632900 -0.17441600

175 C -4.22982700 -1.64797700 -0.07775000 C -5.53203900 -2.46504800 -0.11711100 O -6.58149900 -1.78069800 -0.01083400 O -5.41363300 -3.70128800 -0.24869600 H 4.02646600 4.43550800 -0.48446000 H 4.71197500 2.09350100 -0.68663600 H 1.62464600 4.98340800 -0.01771500 H 0.57971800 -0.34042100 -0.12702400 H -2.75569800 3.90373000 0.42475900 H -5.04050700 3.24356700 0.44828600 H -5.36393700 0.00503200 0.12918100 H 5.28770700 0.21787000 -0.21747200 H 4.82390500 -0.23357500 -1.84686600 H 6.07093200 -2.02230900 -0.71684200 H 4.53029900 -3.08327700 -2.10683800 H 4.26996200 -3.73921300 -0.56191800 H 3.23807800 -2.56984400 -1.15024700 H -2.85810800 -3.24471000 -0.28710200

SCF energy: -1537.395625 Hartree zero-point correction: + 0.325114 Hartree enthalpy correction: + 0.351888 Hartree free energy correction: + 0.266955 Hartree

First five vibrational frequencies (cm-1): 16.81, 21.20, 32.79, 38.92, 54.24

H2Xa, Doublet 0 2 C 3.34533100 3.66746000 -0.12521600 C 3.71934900 2.33449100 -0.28233800 C 2.00600300 4.00125000 0.01583600 C 2.77690600 1.30275300 -0.31591700 C 1.05577200 2.99260800 -0.01231100 C 1.40161400 1.63966300 -0.18551700 C 3.20359800 -0.10206400 -0.49913200 N 0.38419500 0.71534200 -0.20701100 O -0.25347100 3.36952000 0.13533200 C -1.23423100 2.43831700 0.12178900 C -0.92024200 1.07185000 -0.05437800 C -2.54135700 2.88504700 0.28178500 C -1.96848900 0.11709300 -0.07001600

176 C -3.57055800 1.96577500 0.26776300 C -3.28575400 0.57336400 0.09339500 O -4.86916400 2.26925600 0.40767500 N -4.31053600 -0.31091800 0.08554500 C -1.71222200 -1.31896300 -0.25194700 O 2.37630200 -0.99915200 -0.65082300 C 4.68187900 -0.43841900 -0.52969600 C 4.96866500 -1.90670700 -0.24727000 N 4.34714200 -2.83626100 -1.25279400 C 4.49651900 -2.40232900 1.16198500 O 4.14076000 -3.60776600 1.19360300 O 4.56136100 -1.57171600 2.08538300 O -0.55319200 -1.75902300 -0.40726500 C -2.87814000 -2.16193300 -0.23964100 C -4.13035200 -1.64843000 -0.07502300 C -5.43424500 -2.46732200 -0.05173300 O -6.46873000 -1.77459800 0.11343600 O -5.31726700 -3.69908100 -0.19709600 H 4.09962700 4.44233000 -0.10906700 H 4.76869600 2.10264600 -0.38882100 H 1.68048700 5.02469100 0.14773100 H 0.58244500 -0.28930400 -0.34832700 H -2.72853900 3.94304900 0.41187000 H -5.00897300 3.21965200 0.51621900 H -5.27899900 -0.01502700 0.20106200 H 5.21746100 0.13687700 0.22279000 H 5.08381700 -0.13978300 -1.50460900 H 6.04548200 -2.07126700 -0.29835400 H 4.88106800 -2.93936300 -2.11484100 H 4.26001400 -3.73512300 -0.74732100 H 3.39616600 -2.50888900 -1.45871100 H -2.75743700 -3.22817700 -0.36625400

SCF energy: -1537.219971 Hartree zero-point correction: + 0.326376 Hartree enthalpy correction: + 0.352789 Hartree free energy correction: + 0.268660 Hartree

First five vibrational frequencies (cm-1): 16.88, 27.52, 39.04, 44.55, 60.37

A2.6. Coordinates and energies of the leaving groups

177 NH3 0 1 N 0.00000500 -0.00012300 -0.11424100 H -0.80710800 -0.48564500 0.26667400 H 0.82460900 -0.45530800 0.26668400 H -0.01753800 0.94181500 0.26633100

SCF energy: -56.589842 Hartree zero-point correction: + 0.034170 Hartree enthalpy correction: + 0.037982 Hartree free energy correction: + 0.015098 Hartree

First five vibrational frequencies (cm-1): 1060.71, 1650.27, 1650.67, 3469.76, 3583.69

+ NH4 1 1 N 0.00000000 0.00000000 0.00000000 H 0.58983400 0.58983400 0.58983400 H -0.58983400 -0.58983400 0.58983400 H 0.58983400 -0.58983400 -0.58983400 H -0.58983400 0.58983400 -0.58983400

SCF energy: -57.038069 Hartree zero-point correction: + 0.049875 Hartree enthalpy correction: + 0.053672 Hartree free energy correction: + 0.032579 Hartree

First five vibrational frequencies (cm-1): 1476.75, 1476.75, 1476.75, 1725.17, 1725.17

178