Chapter 7: Fluorescence control in natural green fluorescent protein (GFP)-based photonic structures of reef corals – Optical Biomimetics

7

Fluorescence control in natural green fluorescent protein (GFP)-based photonic structures of reef corals

A. SALIH,     University of Western Sydney, Australia

Abstract:

The green fluorescent protein (GFP) and a variety of GFP-like homologues that colour tissues of many reef organisms have revolutionized biological and biomedical research by providing the means to fluorescently tag and visualize the activity of genes and proteins in living cells. This chapter describes how we can further capitalize on what nature has produced by using the GFP group of photoactive proteins, which evolved to perform a variety of biological functions, to develop a range of biomimetic advanced biophotonic applications. The evolutionary pressures that led to the origin of GFP-like fluorescent proteins in marine organisms can be explored in designing novel biomedical sensors, solar cells, biomolecule-based materials and optoelectronic devices. As GFP-like proteins are genetically encodable, this science is posed on the brink of a new technological revolution – to create the means for interfacing biology with electronics, so that devices not only generate energy, but also diagnose diseases and detect pathogens in vivo.

Key words

green fluorescent protein (GFP)

photoactive fluorescent protein (PAFP)

solar energy

photonic

Forster resonance energy transfer (FRET)

biomaterials

7.1 Introduction

Fluorescence is a characteristic of many biological molecules and materials, such as various body fluids, including blood and urine, chitin, collagen, NADH, NADPH, FADH, flavins, phycobiliproteins, chlorophyll and numerous fluorescent dyes. The green fluorescent protein (GFP) is the most widely used fluorescent molecule in cell biology and biomedicine – a mainstay in life-science research. Its popularity is linked to the fact that it is relatively non-toxic and is the only protein in which fluorescent properties are fully genetically encoded and require no other cofactors, other than O2, for their expression (Chalfie 1995). Thus, this molecule is a portable code that can be integrated into the genome of any organism (Fig. 7.1). GFP can be specifically fused to a protein of interest and expression in a diverse range of live cell types, tissues and organisms (Patterson 2007). The emitted fluorescence can then be imaged directly, using fluorescence techniques, such as confocal microscopy, without the requirement of fixation or staining labels. In 2003, the Protein Data Bank (PDB) featured GFP as the molecule of the month, with 31 recorded GFP structures and the number rapidly increased to 266 by 2010 (Ong et al. 2011).

7.1 Structure of GFP-like proteins, showing the 11 β-strands (ribbons) with interconnecting loops of the β-barrel, with the chromophore suspended from an α-helix at the centre of the barrel, where it is well protected from the external environment: (a, b) monomeric form, such as wt-GFP; (c) natural or mutated dimeric form (e.g. HcRed or Renilla GFP); (d) and a tetrameric form of most anthozoan GFP-like proteins (e.g. DsRed, EosFP, Kaede) (adapted from Prescott and Salih 2008).

Although GFP was first isolated in the 1960s from the bioluminescent jellyfish Aequorea victoria (i.e. aeGFP or wild type, wtGFP) its wide-ranging utility in the life-sciences was not demonstrated until the early 1990s (Shimomura 2005). Indeed, it was first thought of as an annoying contaminant to be carefully separated away from aequorin (Shimomura et al. 1980; Tsien 2010). The revolution of the GFP technology started with the cloning of the wtGFP’s cDNA by Prasher et al. (1992), followed by its heterologous expression by Chalfie et al. (1994). These breakthroughs were followed by the rapid explosion of GFP-based applications and culminated in the 2008 Nobel Prize in Chemistry award for the discovery and development of GFP to O. Shimomura (Marine Biological Laboratory, Woods Hole, MA), M. Chalfie (Columbia University, New York, NY) and R. Tsien (Howard Hughes Medical Institute, University of California, San Diego). GFP and related GFP-like proteins continue to be central to a vast array of studies of gene expression and protein tagging and provide novel applications for a variety of sciences.

A lesser-known fact is that GFP-like proteins are the source of colour for a majority of reef corals and many of their relatives – sea anemones, zoanthids, soft corals, sea pansies, etc. (Matz et al. 1999; Salih et al. 2000; Dove et al. 2001; Ando et al. 2002; Labas et al. 2002; Kelmanson and Matz 2003; Wiedenmann et al. 2004; Field et al. 2006; Alieva et al. 2008). The discovery of GFP-like proteins in non-bioluminescent marine organisms was made by Matz et al. (1999) in Lukyanov’s laboratory when the group isolated a gene encoding the red fluorescent protein named DsRed from Discosoma sp, an anemone-like organism closely related to corals, taken from a private aquarium in Moscow. Since then, over 150 genes have been cloned from marine organisms. We now know that the striking colours, which range from bright blue, green and red fluorescent hues (see colour plate section between pages 96 and 97, Plate XX), to the deep pinks and purples of coral branch tips and anemone tentacle tips, are attributable to a super-family of proteins, variously related to the prototypical wtGFP. GFP-like proteins have also been found in evolutionary distant organisms from cnidarians – in crustaceans (copepod, Pontellina plumata) and even a primitive chordate, amphioxus (Shagin et al. 2004; Evdokimov et al. 2006; Bomati et al. 2009; Hunt et al. 2010). In the last decade, these new fluorescent proteins (FPs), especially those derived from reef corals and anemones, have added to the colour palette of GFP-based labels and biosensors and are overtaking in popularity the wtGFP in imaging applications.

Plate XX Diverse colours of GFP-like proteins in reef corals and in biophotonic applications. (a) Patterning of tissues pigmented with GFP-like proteins in different coral species observed in white light and(b) by blue light excitation in the dark, revealing tissue fluorescence. (c) Multicoloured GFPs, including EosFP, in ectodermal cells of the coral Lobophyllia hemprichii. (d) Cloned and purified cyan, green and red GFPs from the coral Acropora millepora. (e) The excitation and emission spectra (only 3 of 5 GFPs are shown) of these cyan, green and red proteins in coral tissues exhibit characteristics of donor– acceptor pairs tuned for fluorescence energy transfer, with emissions from cyan spectrally overlapping the excitation of the green emitters and the emission of greens overlapping the excitation of reds. Red spectra and shading in (e) show excitation and emission of photosynthetic pigments of coral’s symbiotic microalgae. Energy transfer via GFPs channel excess energy towards peridinin, the accessory photosynthetic pigment of microalgae and into the gap of photosynthetic absorption (adapted from Salih et al. 2004). (f) Green- to-red photoconversion of EosFP from L. hemprichii showing absorption and emission spectra of the green and photoconverted red forms (adapted from Wiedenmann et al. 2004). (g) mEosFP expressed in a mammalian cell and photoconverted to red by a brief irradiation by 405 nm (Salih, unpublished). (h) Encoding in four colours using IrisFP in a surface of a large agarose-Ni-NTA bead (117 mm diameter) imaged at 488 nm (52 W/cm2) and 561 nm (125 W/cm2) for green and red channels (sub-panels i–viii). Using a 405-nm laser light, the letters were written, fully or partially photoconverting the initially green molecules to their red emitting states and creating red, yellow and green colours (adapted from Adam et al. 2010).

The evolutionary pressures on coral reefs have forced this natural bio-optical system to become highly optimized. By developing an understanding of how GFPs function in coral biology, the knowledge can serve as a guide for the rational design of advanced biomimetic solutions to develop protein-based optical sensors and materials with improved functionalities. To understand how we may develop biomimetic devices by copying what nature has produced, this chapter first briefly describes the molecular structure and spectral diversity of GFP-like proteins, followed by the discussion of some of their biological functions in marine organisms. Although a full description of the wide-ranging applications of GFP-like proteins can only be scratched at here, several new directions are outlined, and future developments are discussed to set the stage for the coming generation of GFP- based advanced technologies.

7.2 Green fluorescent protein (GFP) structure and diversity

The wtGFP and other GFP-like proteins all have an 11-stranded β-barrel, with a diameter of about 24 A° (~2 nm) and a height of 42 A° (~3 nm), with an α-helix running through the β-barrel (Fig. 7.1) (Prasher et al. 1992; Ormo et al. 1996). A p-hydroxy-benzylideneimidazolidinone chromophore (Fig. 7.2) is located in the centre of the barrel in the majority, but not in all GFP-like proteins, and is protected from solvents by lids composed of short helices. The chromophore is formed by the cyclization of the adjacent Ser65-Tyr66-Gly67 (the number denotes the position in the intact peptide sequence) residues within this hexapeptide sequence (Cody et al. 1993). Aside from marine organisms, a GFP-like folding topology consisting of the 11-strand β-barrel domain, has been identified even in human extracellular matrix proteins. This protein, termed G2F (globular-2) fragment, is part of a multidomain protein and provides a binding surface for protein–protein interactions (Hopf et al. 2001).

7.2 Steps in the formation of the A. victoria wtGFP chromophore (from Day and Davidson 2009).

Thus, the kingdom Metazoa has two gene lineages within the GFP protein superfamily: the colourless G2F lineage and the GFP-like lineage. The two branches appear to have originated from a common ancestor predating the divergence of Radiata (phylum Cnidaria) and Bilatera (phyla Arthropoda, Mollusca, Chordata, etc.) (Matz et al. 2006).

The formation of wtGFP chromophore and protein maturation has been the focus of research by several groups and can be summarized as a cyclization–oxidation–dehydration sequence of events, where the oxidation step is solely guided by main chain chemistry (Fig. 7.2) (Rosenow et al. 2004; Zhang et al. 2006; Day and Davidson 2009; Wachter et al. 2010). Molecular oxygen is crucial for the development of the chromophore’s fluorescence and at least one oxygen molecule is required for dehydrogenation (Tsien 1998). Chromophore maturation results in a highly conjugated pi-electron resonance system responsible for the protein’s spectroscopic and photo-physical properties. The mechanism of maturation of the red FPs, such as DsRed, has been revised several times and is excellently reviewed by Wachter et al. (2010). The generation of the DsRed chromophore does not depend on light exposure and is formed by an oxidation reaction that extends the conjugated system, forming a red shifted absorbance and fluorescence (Baird et al. 2000; Gross et al. 2000; Wachter et al. 2010).

The similarity of the amino acid sequences of the GFP-like proteins with wtGFP from A. victoria ranges from 90% to less than 20%. In wtGFP and its variants the β-barrel is a cylinder, while the GFP-like proteins of anthozoans have an elliptic shape (Baird et al. 2000; Yarbrough et al. 2001). Even though these proteins are derived from completely unrelated species and sometimes have little homology, they exhibit very little variation in their tertiary structure. The cylindrical geometry of GFP appears to function in protecting the chromophore, making it remarkably photostable and bright, resistant to reactive oxygen, light, pH and many denaturing agents (Tsien 1998). The term ‘light in a can’ has been applied to GFP and related proteins because of these protective properties of the protein shell.

The completely autocatalytic formation of the light-emitting chromophore makes it possible for GFP to be expressed in the genetic fusions with other proteins for studying their expression, localization and function inside living cells and whole organisms. Thus, the main current applications of GFP-like proteins involve their use in imaging as fluorescent markers in single- and multicolour applications. The need simultaneously to visualize multiple proteins drives the demand for additional FP spectral variants. The wtGFP was mutated to produce variants emitting in the blue (BFP), cyan (CFP) and yellow (YFP) colours (reviewed by Tsien 1998, Tsien 2010), but orange and red mutants were not developed until GFPs were discovered in anthozoans (Matz et al. 1999).

The fluorescence colour repertoire of anthozoan GFPs is significantly broader than that of the bioluminescent organisms (Plate XX): cyan FPs with emissions at 470–499 nm; green FPs with emissions at 500–525 nm; yellow FPs with emissions at 526–545 nm; and orange and red FPs covering the rest of the visible spectrum, up to 640 nm (Matz et al. 1999; Ando et al. 2002; Kelmanson and Matz 2003; Chudakov et al. 2004; Wiedenmann et al. 2004; Alieva et al. 2008). DsRed alone led to the development of 14 GFP-like protein colour variants (Shaner et al. 2004). Far red fluorescent proteins are in high demand for in vivo whole body imaging; however, there has been no naturally derived colour variants above 680–700 nm discovered so far and much effort is spent on mutagenesis of red FPs, with some successful development of near-infrared variants – Katusha, eqFP650 and eqFP670 that emit at 640 to 670 nm (Shcherbo et al. 2007, 2010; Lukyanov et al. 2010).

Anthozoans also possess GFP-like chromoproteins (CPs) that are brightly coloured blue, pink or purple molecules that do not fluoresce (Dove et al. 1995; Takabayashi et al. 1995; Dove et al. 2001; Alieva et al. 2008). Several of these proteins are photoactive and can spontaneously acquire red fluorescence under green light irradiation (Lukyanov et al. 2000Lukyanov et al., 2005; Chudakov et al. 2003; Salih et al. 2006b; Prescott and Salih 2009) and this property is referred to as kindling (Lukyanov et al. 2000). Moreover, coral-derived GFPs possess other unusual photoactive characteristics, described below, that make them highly useful in bioimaging and biotechnological applications.

The majority of GFP-like proteins from non-bioluminescent organisms discovered to date display variable degrees of quaternary structure. They are composed of monomeric subunits with blue, green or red emitting chromophores (Baird et al. 2000). The monomers show a similar topology to GFP, but form tetrameric (Fig. 7.1(d)) or form obligate oligomeric structures and aggregates both in vivo and in vitro (Baird et al. 2000; Wall et al. 2000; Yarbrough et al. 2001). The monomers do not act independently from each other but are energy coupled via a Förster resonance energy transfer (FRET) mechanism (Förster 1948). When a tetrameric protein is excited by blue light irradiation, the observed red emission is not due to the direct excitation of the red chromophore, but results from the excitation transfer from the green to the red emitting fluorophore (Baird et al. 2000; Wall et al. 2000; Yarbrough et al. 2001). In fact, the property of tetramerization and oligomerization appears to be a unifying feature of the majority of FPs from non-bioluminescent organisms. Alternately, GFPs in bioluminescent organisms, such as Aequorea and Renilla, form hetero-tetramers with their respective photoproteins.

Two main mechanisms for protein aggregation have been proposed. One involves the non-specific interactions of ‘sticky’ hydrophobic patches on the molecular surface (Eaton and Hofrichter 1990). The other mechanism involves the electrostatic interactions between positively and negatively charged surfaces. Oligomerization of DsRed occurs at two chemically distinct protein interfaces to assemble the tetramer (Baird et al. 2000; Wall et al. 2000). However, it does not contain the pronounced hydrophobic areas that may cause strong interactions between tetramers. Based on computer calculation of the electrostatic potential of tetrameric DsRed, it was revealed that the protein surface is mostly negatively charged, and it was proposed that each tetramer is able to form up to four salt bridges with adjacent tetramers, resulting in the net-like polymeric structure (Yanushevich et al. 2002).

Oligomerization can cause many problems when FPs are used as in vivo imaging probes, particularly when fused to other proteins of interest, disrupting normal protein distribution and function and can critically affect cellular processes. On the other hand, the tetramerization and oligomerization of FPs presents a remarkable opportunity for the development of photostable and photoactive biopolymers or hybrid organic materials, since the quaternary structure makes the proteins highly photostable and can provide optimized means for efficient energy and electron transfer between the monomeric subunits.

7.3 Photoactive fluorescent proteins (PAFPs)

During the last decade, a remarkable group of GFP-like proteins has been discovered that can spontaneously change their optical properties in response to irradiation (Lukyanov et al. 2000; Ando et al. 2002; Patterson and Lippincott-Schwartz 2002; Chudakov et al. 2003; Wiedenmann et al. 2004; Lukyanov et al. 2005; Remington, 2006). They are referred to as photoactive fluorescent proteins (PAFPs), optical high-lighters, photoconverting or photoswitching proteins (Lukyanov et al. 2005; Patterson and Lippincott-Schwartz 2002; Lippincott-Schwartz and Patterson 2008). They all have the general characteristic that they are initially dark at the pre-activated fluorescence wavelength but upon activation display an increase in fluorescence and are ‘highlighted’ over a darker background, such as kindling (KFP) and Dronpa proteins from anthozoans (Chudakov et al. 2003; Habuchi et al. 2005). Other such proteins change colour upon activation, from cyan to green (Chudakov et al. 2004); from green to red (e.g. Ando et al. 2002; Wiedenmann et al. 2004) (see Plate XX); from red to green; and from red to yellow (Salih et al. 2006b). Yet others have both photoconversion and photoswitching properties, such as IrisFP (Fig. 7.3), which was developed from EosFP (Adam et al. 2008).

7.3 Reversible photoswitching of green and red forms of IrisFP in crystals. (a) The green form in a capillary was illuminated with 488 nm light, while 405 nm light was switched on every 7.5 s for 25 ms. (b) After green to red photoconversion of IrisFP crystal by strong irradiation at 405 nm was achieved its emissions were imaged after continuous illumination with 532 nm light and irradiation by 440 nm light switched on and off every 7.5 s for 200 ms. Inserts show representations of the β-barrel structure of IrisFP and structural models of its chromophore in its two bright and two dark states (adapted from Adam et al. 2010).

In live cell imaging PAFPs are particularly useful quantitatively to investigate the dynamics of proteins within a population in cells or tissues. They can be used directly to highlight or photoconvert a pool of molecules by the irradiation of a selected area of interest and then to follow the signal of the photoactivated population with high signal to noise ratio (e.g. Lippincott-Schwartz et al. 2001; Lukyanov et al. 2005; Prescott and Salih 2009).

The green-to-red photoconverting PAFPs appear to be especially prominent in numerous cell imaging applications and two such proteins, Kaede from the coral Trachyphyllia geoffroyi (Ando et al. 2002) and EosFP from the coral Lobophyllia corymbosa (Plate XX) (Wiedenmann et al. 2004), have become popular cellular probes. They can be photoactivated at irradiation by near UV light (~ 400 nm) and change colour from the pre-activated green fluorescence (emission at 516–518 nm) to a new red fluorescence (emission peak at 580–581 nm) (Plate XX). EosFP has been mutated into two dimeric forms, d1EosFP and d2EosFP, and a monomeric mEosFP (Wiedenmann et al. 2004). The shift in both the excitation and the emission peaks following photoconversion results in a large contrast with the background after photoactivation at more than 2000-fold increase in the red-to-green fluorescence ratio (Wiedenmann et al. 2004).

Kaede and EosFP, as well as other green-to-red PAFPs so far reported (e.g. IrisFP, Dendra2, KikGR, LcorFP, etc.) contain a chromophore derived from the tripeptide His-Tyr-Gly (HYG) (Mizuno et al. 2003). It initially emits green fluorescence in which UV wavelengths irradiation produces a cleavage between the amide nitrogen and α-carbon atoms in the histidine residue, leading to an extension of the chromophore conjugation to the histidine side chain and a spectacular transformation into bright red fluorescence (Mizuno et al. 2003).

Another group of PAFPs, belonging to the CP protein group described in the previous section, also contain the kindling fluorescent proteins (KFPs). These naturally occurring proteins have the basic β-barrel structure of wt-GFP and form the internal chromophore inside the β-barrel. They are less than 30% homologous to wtGFP and are non-fluorescent prior to photoactivation, but following a brief irradiation by green wavelengths spontaneously acquire bright red fluorescence (Lukyanov et al. 2000; Chudakov et al. 2003; Salih et al. 2006b). The first KFP was discovered in the purple tentacle tips of the sea anemone, Anemonia sulcata and absorbs light at 568 nm and after irradiation fluoresces at 595 nm (Lukyanov et al. 2000). Kindling is a reversible process as the fluorescent state gradually relaxes back to a non-fluorescent state in the dark at partial kindling (Lukyanov et al. 2000; Chudakov et al. 2003). The bright form can be instantly switched off by a flash of blue light and the switching process can be repeatedly cycled. The high-resolution crystal structure of KFP revealed that in the dark-adapted state the protein has a distorted chromophore in the trans-noncoplanar configuration and following light activation the chromophore undergoes cis–trans isomerization to a fluorescent state (Andresen et al. 2005; Quillin et al. 2005). By comparison, most FPs have chromophores that are cis-coplanar.

The diverse opportunities presented by the photoactive properties of these GFP-like proteins are only beginning to be explored and they provide an excellent foundation upon which to develop optical biomimetic technologies, some of which will be described in the sections below. In summary, the photoconversion mechanisms of PAFPs are not yet fully understood at the molecular level but it is known that they involve photo-induced isomerizations, such as cis-trans transformation, changes in protein microenvironment, other structural changes, such as bond breaking, proton and charge transfer between the protein and the chromophore.

7.3.1 Biological functions of GFP-like proteins

In bioluminescent jellyfish, wtGFP has existed for more than one hundred and sixty million years. It is localized in several hundred photo-organs in tissues on the umbrella margin, which when stimulated give off green light (Plate XX). GFP is involved in an efficient Forster resonance energy transfer (FRET) (Forster 1948) as an acceptor protein, transforming blue light (470 nm) emitted by the luminescent donor aequorin into green light (508 nm) (Morin and Hastings 1971; Morise et al. 1974). However, the biological relevance of this process to the organism and in fact, within other bioluminescent organisms, is still unknown.

Indeed, the function of the enormous variety of GFP-like proteins found in the non-bioluminescent cnidarians, such as corals and other anthozoans, is only beginning to be understood (Kawaguti 1944; Catala 1959; Matz et al. 1999,Matz et al. 2006; Salih et al. 2000; Dove et al. 2001,Dove et al. 2008; Mazel et al. 2003; Wiedenmann et al. 2004; Field et al. 2006; Kao et al. 2007; Oswald et al. 2007; Gruber et al. 2008). As anthozoans have few predators, do not depend on mate attraction for reproduction and do not need to attract their prey, the primary function of GFPs cannot be involved in defensive light emission, reproductive signalling, prey lures and in food assimilation. One plausible hypothesis is that some tissue fluorescent patterns of symbiotic anthozoans may serve as attractants to symbionts, which have been shown to be attracted by green light (Hollingsworth et al. 2005). Since free-swimming symbiotic algae may be taken up from the water column throughout the host’s life-history, the brightly fluorescent colours patterning the area around the mouth may serve as an attractant. Many of the known and hypothesized functions of GFP-like proteins are linked to light and many proteins are in fact, photoactive, as was discussed in the previous section of this chapter, and can spontaneously alter their optical properties in response to irradiation.

The sunscreening and light modulatory roles of GFP-like proteins are the most experimentally explored functions of these molecules in marine organisms. An early suggestion of a sunscreening by coral coloration (Kawaguti 1944) was substantiated by later research that provided the first direct evidence that GFP-like proteins function in light-optimization of coral tissue (Schlichter and Fricke 1990), and in shallow corals are photo- protective (Salih et al. 2000, 2001,Salih et al. 2006a;Salih et al. 2006b, Dove et al. 2001, Dove et al. 2008; Gilmore et al. 2003; Roth et al. 2010). Reef corals require effective photoprotection since they are exposed to massive doses of solar radiation in the oligotrophic clear waters of the tropical oceans in which sunlight penetrates to considerable depths (Jerlov 1976; Ackleson 2003).

While often referred to as the ‘rainforest of the ocean’, corals are actually animals, not plants. But they do have plant-like adaptations since one of the most fascinating aspects of their biology is the symbiotic relationship with dinoflagellate microalgae, Symbiodinium microadriacticum (termed zooxanthellae) that live in millions in coral’s endodermal cells. Zooxanthellae carry out photosynthesis and translocate sugars into the coral’s tissues, providing up to ~ 95% of coral’s metabolic requirements (Muscatine et al. 1984). This symbiotic relationship is the reason corals require sunlight and cannot rely on solar blocking molecules, such as melanins, used by other animals. Nevertheless, they have evolved to be incredibly efficient in capturing solar energy and converting it to chemical energy via photosynthesis of their symbionts.

Even under normal environmental conditions, and even more than many photosynthetic organisms, corals are routinely exposed to very variable illumination conditions that drastically change due to diurnal variations. Underwater, wave lensing effects can create powerful light flashes of extremely high intensity (Veal et al. 2010). Recent studies showed that these intensities often reach above 150% of mean solar irradiance even at a depth of 20 m at a coral reef (Veal et al. 2010).

The presence of photosynthesizing organisms in coral tissues presents a major threat, since high levels of the resultant photo-oxidants can cause cellular damage and death. The whitening of coral tissues (referred to as coral bleaching) during conditions of adverse stress owing to high solar radiation, warming of seawater or other stressful events, is primarily caused by the loss of photosynthetic pigments and of zooxanthellae (Brown 1997). During the last two decades, coral bleaching has begun to occur on massive scales, causing many kilometres of reefs to bleach and the phenomenon has been linked to global climatic changes (Hoegh-Guldberg 1999). The mechanism of coral bleaching has been linked to the build-up of photo-oxidative stress, photoinhibition of the microalgal photosynthesis, damage to Photo- system II (PSII), increased generation of reactive oxygen species (ROS), damage to membranes, DNA and various cellular organelles in both the symbionts and the coral host (Weiss 2008). The photodamage is exacerbated at light intensities below those normally required to saturate photosynthesis (Asada 2006) when other stressful environmental or man-made factors limit the ability of zooxanthellae to acquire and assimilate carbon dioxide (CO2). As the seawater warms due to climate change and temperatures increase above the normal summer maxima, the zooxanthellae living in symbiotic harmony with their coral hosts become damaged and expelled, leaving the starkly white, bleached coral reefs. In both the zooxanthellae and the coral tissue the various alternative sinks for electrons become turned on to prevent photo-oxidative damage (Lesser 2007; Weiss 2008).

Accumulating scientific evidence shows that GFP-like proteins have evolved to regulate the solar radiation impacting on the coral and other marine organisms, particularly those that are symbiotic with photosynthetic zooxanthellae. But even organisms that lack symbionts may experience photo-oxidative stress as a result of the fluctuating light environments encountered underwater and may rely on GFPs for light modulation to protect tissues from photodamage (Kahng and Salih 2005). In corals, experimental studies provide a strong evidence that GFP-like proteins form a highly dynamic light modulation system that can precisely alter the intracellular light environment while effectively harvesting light energy and controlling the ensuing negative consequences of living in a high-light environment. The mechanism of photoprotection by GFPs is highly sophisticated and involves light absorption, scattering and light energy transformation (Salih et al. 2000,Salih et al. 2006b; Dove et al. 2001; Gilmore et al. 2003; Cox et al. 2007).

Although sunscreening and light modulation are clearly important functions of GFP-like proteins, they are most certainly not the only functions. Importantly, it has been shown that FPs can directly absorb the reactive oxygen species (ROS), including H2O2 (Bou-Abdallah et al. 2006) and can function as antioxidants, protecting corals from the excessive levels of ROS generated in their cells during high-light stress (Palmer et al. 2009). Thus, under stressful conditions of globally changing climate, FPs can enhance the survival of pigmented corals by both sunscreening and by directly reducing the build-up of ROS by quenching reactive oxygen species (Bou-Abdallah et al. 2006).

Recently, a group led by K. Lukyanov made a ground-breaking discovery that upon exposure to blue light, green GFP-like proteins donate or accept electrons from biologically relevant donor or acceptor molecules and turn red (excitation 575 nm and emission 607 nm) as a result (Bogdanov et al. 2009). The process was referred to as redding or reddening, and occurred after only a brief irradiation. The mechanism is still not fully understood and is being intensely investigated. It was hypothesized to produce a stable radical and, since the green fluorescence disappeared faster than the red fluorescence increased, reddening was deduced to be a two-step process (Bogdanov et al. 2009).

Unlike electron transfer and reddening under unaerobic conditions, known to occur in GFP for some time, this newly discovered process proceeded under aeroboic conditions and revealed an exciting possibility that the primary function of GFPs is in photoreduction. The light-prompted passing of electrons by GFPs was referred to as the discovery of ‘animal photosythesis’ by the media. Coral FPs also undergo reddening in vivo in the presence of electron acceptors and their light-induced electron transfer properties are especially prominent in certain cells or organelles of coral tissues – mitochondria, cells undergoing rapid growth and, interestingly, in neurons or cells with sensory functions (Salih and Geny, unpublished). The electron transport properties appear to be key natural functions of GFP- like proteins in corals and other marine organisms.

The excited state proton transport (ESPT) is another exciting major function of the GFP-like proteins that is emerging as a result of research into protein photochemistry, but which has not yet been experimentally confirmed to occur in vivo in marine organisms. Proton transport is one of the most fundamental processes in biology as energy from light or respiration is used to generate a trans-membrane proton gradient. Usually, this is achieved by membrane-spanning enzymes known as ‘proton pumps’. ESPT plays a key role in the biochemical processes that convert sugar to a form that cells can use – adenosine triphosphate (ATP). Cellular proton concentration changes can store this energy and the production of ATP from sunlight also involves proton transport processes.

Moreover, proton transport plays a major role in the photochemistry and activation of many receptors – the visual and the sensory rhodopsins (retinal proteins), proteorhodopsin, phytochromes and phototropin (e.g. Losi 2004; van der Horst and Hellingwerf 2004). The prerequisite for signal transduction is charge displacement within a sensory protein or protonation changes that can induce the conformational changes. The red light photoreceptors in plants, the phytochromes, for example, regulate many developmental processes such as flowering and shade avoidance (Franklin and Whitelam 2007). Another example of a proton transferring protein is the photoactive yellow protein (PYP), which is a water-soluble molecule, involved in sensing a wide range of stimuli, including light, oxygen, redox potential, and a variety of ligants (Imamoto and Kataoka 2007; Wachter and Remington 1999).

A major discovery was made when it was found that wtGFP’s p- hydroxybenzylidene-imidazolidinone chromophore acts as a photoacid and light triggers an ultrafast ESPT (Chattoraj et al. 1996; Agmon 2005). The protein has two distinct absorption bands at 396 and 476 nm owing to the protonated (neutral, state A) and deprotonated (anionic, state B) forms of the chromophore (Heim et al. 1994; Chattoraj et al. 1996). While light is absorbed by both forms, fluorescence occurs from the anionic form, state B. The conversion of the neutral state A, which emits blue fluorescence, to the anionic state B, which emits green fluorescence, occurs by the excited- state ultrafast Tyr66 phenolic proton shuttling through an extensive hydrogen-bonding network (Chattoraj et al. 1996; Meech 2009). The change from forms A to an intermediate form I is solely a protonation change, while the change from state I to B is a conformational change (Stoner-Ma et al. 2005).

The X-ray structural studies of wtGFP (Yang et al. 1996; Ormo et al. 1996; van Thor et al. 2005) found that the active site proton wire (i.e. forming chains of water molecules and protonatable amino acids) is very extensive, originating at Tyr66 in the chromophore and beyond it to Glu5 and other groups on the protein surface (Ormo et al. 1996; van Thor et al. 2005). Ultrafast time-resolved IR spectroscopy confirmed the proton pathway and found it to be reversible, constituting a tiny ‘photocycle’ (Stoner-Ma et al. 2005). Another proton wire in GFP was also recorded (Shinobu and Agmon 2009).

Thus, in wtGFP, illumination initiates a proton transfer through a proton wire or several wires, formed by the chromophore acting as the proton donor, via a water molecule W22, Ser205 and Glu222 (the acceptor), on a picosecond time scale, with subsequent longer distance transfers occurring at nanosecond scales. Research of GFP-based proton wires is being investigated in a number of GFP-like proteins and by a variety of techniques – molecular research, X-ray protein structural determination, ultrafast fluorescence and time-resolved vibrational spectroscopy, complemented by quantum chemical calculations.

The biological function of GFP-based proton pumping and proton wires in marine organisms has only been speculated upon. In various biological systems, proton wires are made up of hydrogen bonding networks within the interior of proteins, consisting of predominantly oxygen and nitrogen atoms of amino acid side chains and the migrating protons use these atoms as stepping stones. As in other organisms, proton wires have prominent roles in trans-membrane proton transport and may perform this function in anthozoan tissues. It has been also suggested that GFPs may function as portable proton pumps within cnidarian cell cytoplasm (Agmon 2005). Thus, proton pumping properties of GFPs may perform diverse biological functions in marine organisms – photosensing, chemosensing, production of ATP (or sugar) from sunlight, calcification in corals, etc. and is a field of science that is becoming to be the focus of researchers worldwide.

7.3.2 GFP-like proteins as solar energy transfer arrays

The extraordinary adaptations for solar energy modulation and channelling by multicoloured GFP-like proteins in coral tissues present a unique biomimetic opportunity. The multitudes of anthozoan tissue patterns are attributable to diverse GFP-like proteins, many of which have different covalent structures of the protein chromophores and with different arrangements of their molecular environments. GFP-like proteins are localized in a variety of cellular and subcellular compartments in corals: in cells of the epidermal and endodermal tissue layers; in granules of fluoro-chromatophores; intracellularly, within organelles such as nucleus, mitochondria, endoplasmic reticulum, Golgi, in intracellular vesicles, cytoplasm and even within membranes (Plate XX) (Mazel 1995; Salih et al. 1998,Salih et al. 2000,Salih et al. 2006a; Cox and Salih 2006).

Surprisingly, it was discovered that the majority of corals usually have spectrally heterogeneous mixtures of GFP-like proteins (Plate XXe) within their cells – two or three, and often as many as four to six, and occasionally even more, can occur in a single cell. For example, four principal colours – cyan, green, red and non-fluorescent blue FPs – were cloned from the coral Acropora millepora and six molecularly distinct GFP-like proteins were found responsible for these colours (Cox et al. 2007; Alieva et al. 2008). The proteins formed heterogeneous mixtures within individual cells when live A. millepora tissues were analysed by confocal microscopy with microspectral detection (Salih et al. 2000, 2003, 2006a,b; Cox et al. 2007). They exhibited spectral characteristics of donor–acceptor pairs tuned for fluorescence energy transfer, with emissions from cyan FPs spectrally overlapping the excitation of the green emitters and the emission of greens overlapping the excitation of the reds (Plate XXe). The analysis of spectral properties of many coral species showed similar arrangements (e.g. Kelmanson and Matz 2003), with emissions of one FP precisely overlapping the excitation spectrum of the neighbouring FP, and forming progressively red shifted donor–acceptor assemblies tuned for the transfer of solar energy.

The mechanisms of energy transfer between these spectral pairs were found to be due to both the radiative and non-radiative FRET processes (Salih et al. 2000; Gilmore et al. 2003; Cox et al. 2006; Roth et al. 2010). FRET is known as the energy transfer from an excited state of a donorchromophore to an acceptor without the emission of a photon and occurs as a result of a dipole–dipole interaction between the donor and the acceptor (Förster 1946). The fluorescent emission spectrum of the energy donor must overlap the absorption spectrum of the energy acceptor. The FRET efficiency (E) varies as the inverse sixth power of the distance between the two molecules (denoted by r):

The distance at which energy transfer is 50% efficient, i.e. when 50% of the excited donors are deactivated by FRET, is defined by the Förster radius (R0). The magnitude of R0 is dependent on the spectral properties of the donor and the acceptor dyes. FRET, therefore, decreases as the inverse sixth power of the donor–acceptor distance, with typical values of the R0 of ~2–10 nm. The probability of finding a donor and an acceptor within the Förster radius in corals can only be high within intracellular spaces in which the donor–acceptor pairs are localized within the same molecular system. The multichromophoric tetrameric and oligomeric structure of anthozoan GFP-like proteins may have evolved to optimize intramolecular FRET. However, to what degree FRET occurs among multichromophoric aggregates in coral cells is still to be determined.

One explanation for the presence of these energy channelling protein arrays in anthozoan cells is to increase the efficiency of light modulation and photoprotection of coral tissues and photosynthetic symbionts. Solar energy is transferred from protein to protein and excess energy is dissipated away from cellular targets that can be photo-oxidized (Salih 2000; Salih et al. 2000–2004; Gilmore et al. 2003; Cox and Salih 2005). FRET serves as a prominent relaxation channel, especially in multichromophoric systems (Forster 1965). The acceptor can be a fluorescent or a non-fluorescent GFP- type protein. If the acceptor is fluorescent, the transferred energy can be emitted as fluorescence characteristic of the acceptor and if the acceptor is not fluorescent, the energy can be lost through equilibration with the surrounding cytoplasm or heat dissipation via molecular vibrations. The presence of light tuning protein arrays has never been previously found in tissues of marine invertebrates other than in visual systems, certain types of non-visual retinal pigments and in photosynthetic systems. The energy transfer function of GFP-like proteins is emerging to be a true system property of non-bioluminescent marine organisms and raises significant questions regarding the structuring of GFP-like aggregates for effective excitation, energy channelling and possible, other functions, linked to intra- cellular photon and electron transfer.

7.3.3 Coral GFPs as improved FRET and other cellular probes

The use of GFPs as in vivo cellular imaging probes has been covered in many excellent publications and reviews (e.g. Tsien 1998,Tsien 2010; Lippincott- Schwartz et al. 2001; Remington et al. 2004; Verkhusha and Lukyanov 2004; Patterson 2007; Miyawaki 2009; Wu et al. 2011; Stepanenko et al. 2011) and will not be addressed here. These proteins have been successfully expressed in cell cultures and organisms, such as bacteria, yeast, plants, worms, insects and vertebrates (reviewed, Stepanenko et al. 2008). They have been used to study numerous diseases, such as cancer, and the activity of pharmaceutical drugs (e.g. Chishima et al. 1997; Hoffman 2005).

Similarly, the field of GFP-based FRET imaging probes and biosensors, used precisely to measure molecular distances between two fluorophores, is a highly advanced science, with a multitude of probes and sensors that are too numerous to be detailed here. FRET has been used to investigate and map a variety of complex biological structures, such as proteins and macromolecular assemblies including ribosomes and nucleosomes. Imaging using FRET techniques have provided unprecedented tools to quantify molecular dynamics, such as protein–protein interactions, protein–DNA interactions, protein conformational changes, concentration and movement of molecules within physiological environments, determination of their concentrations across cellular compartments, tissues and even organs (e.g. Bastiaens and Pepperkok 2000; Miyawaki and Tsien 2000; Miyawaki et al. 1997; Miyawaki et al. 2003; Miyawaki 2005; Li et al. 2006; Grant et al. 2008; Mank and Griesbeck 2008; Tsutsui et al. 2008).

The use of GFP-like proteins in live cell imaging has many advantages compared to fluorescent dyes and other fluorophores. They are genetically encodable, in most cases are non-toxic or have low toxicity, are very bright and photostable, are highly resistant to most proteases, organic salts, alkaline pH and high temperature and their fusion to a target protein rarely affects the function of that protein (Chudakov et al. 2010). Coral-derived GFP-like proteins are especially bright and often do not bleach even when exposed to high laser irradiation. Since corals live in sunlit and warm tropical seas, their GFP-like proteins are resistant to high irradiances and warm temperatures and are therefore brighter and more temperature tolerant than many (although not all) genetically unmodified GFPs from bioluminescent organisms. Their antioxidant functions in anthozoans may alter some of the cellular activity when they are expressed in cells, but this has rarely presented a problem. Importantly, these GFP-like proteins do not usually generate large quantities of toxic ROS during irradiation.

Given the natural spectral tuning for FRET of many coral GFP-like proteins discussed in the previous section, researchers searching for the best FRET probes would benefit by cloning the donor–acceptor pairs from a single coral, or even better, from a single cell of one coral. Recent research indicates that other marine organisms can be investigated for naturally FRET-tuned GFPs (e.g. Aglyamova 2011). Biomimetic approaches are likely to make major breakthroughs in GFP-based imaging applications, given the newly discovered redox active, electron and proton transferring GFPs.

7.3.4 GFP-based solar energy materials and solar cells

The global worsening environmental pollution and energy shortages have raised the awareness of the need for urgent sustainable development of alternative clean energy technologies. A multitude of green energy projects around the world are competing to design efficient methods to convert solar energy into electricity and replace the dependence on fossil fuels. Since the 1950s, the photovoltaic market has been dominated by solar cells made with wafers of crystalline silicon, known as the first-generation solar cells. However, they are expensive and are uneconomical for mass use. The second generation of solar cells, first developed in the 1970s, known as thin cells, use films of semiconductors, such as cadmium telluride (CdTe) and copper indium selenide (CIS) instead of silicon, making them cheaper to manufacture and there is a considerable effort spent to increase their durability and efficiency.

The third-generation solar cells are organic cells and cells based on biomimetics of photosynthetic light-harvesting (Gust et al. 2001) – the dyesensitized solar cells (DSSCs). The highly evolved system for efficient energy channelling via GFPs in photosynthetic corals can be used as a model to develop advanced DSSCs.

Plant chlorophylls act as active light-harvesting antennae that channel light into photosynthetic centres where sugars and oxygen from carbon dioxide from air and water are formed. The process requires an electron that acts as a powerful reducing agent, reducing molecules of carbon dioxide to form carbohydrates. Only a small fraction of all chlorophylls are directly involved in the process of transformation of an electronically excited state into the primary electrochemical product (Gaffron and Wohl 1936). The majority of pigments are involved in light absorption and efficient transfer of the electronically excited states to pigment molecules where the photochemical reactions take place (Green 2003). The biological electron transfer in photosynthesis occurs over a long range very efficiently through the biomolecules (Deisenhofer 1985). Redox potential differences drive the specific energy and electron transfer events, which are also determined by the electron transfer property of molecules, especially the electron acceptors–donors, electron relay and sensitizer molecules (Karvanos 1993). Plants and certain bacterial systems achieve light-harvesting efficiencies at above 99%. As discussed earlier in the chapter, corals are incredibly efficient in capturing solar energy and converting it to chemical energy via photosynthesis of their symbionts. Although, the in vivo cellular generation of energy in corals by GFPs has only been hypothesized, the efficient light energy transfer among GFP multichromophoric arrays has many similarities with the photosynthetic antennae and these proteins promise to be excellent candidates for DSSC development.

The first DSSC, invented by Michael Grätzel and B. O’Regan in 1991, is known as Grätzel cell. In 2012, the Interdisciplinary Committee of the World Cultural Council awarded Michael Gratzel the Albert Einstein World Award of Science, in recognition for the major contribution of this invention to solar technologies. DSSCs belong to a photo-electrochemical solar cell type that uses two different chemical compounds to harvest light and distribute photoelectrons – microcrystals or nanoparticles of an inorganic n-transporter (e.g. titanium dioxide), coated by a layer of a sensitizing dye (e.g. ruthenium complex). A single coating of a fluorescent dye on the solar cells gives 30% increase in the energy conversion efficiency (η, the percentage of power converted from absorbed light to electrical energy) of the solar cell (Maruyama et al. 1998). Upon light absorption, the sensitizer is promoted into an electronically excited state from where it injects an electron into the conduction band of n-transporter. DSSCs are low-cost materials, can be produced without using toxic chemicals and can be assembled into a wide variety of sizes and shapes using simple processes similar to inkjet printing.

The constituents of the organic dye sensitizer determine to a great extent the efficiency of DSSCs (Gregg and Hanna 2003). An organic dye can be tuned to optimize its performance and combinations of dyes are used to increase the solar collecting efficiency and thereby increase electricity generation (e.g. Maruyama et al. 1998; Zhang and Jenekhe 2000; Hardin et al. 2009). The mechanism of the photocurrent generation differs from that of inorganic solar cells, since the absorption of light leads to bound electron–hole pairs, the excitons (Gregg and Hanna 2003; Hoppe and Sariciftci 2004). The efficiency of dissociation of the bound excitons is enhanced either by a stronger electric field or by FRET processes in the donor–acceptor heterojunctions, leading to a fast charge transfer across the interface (Sariciftci et al. 1992; Liu et al. 2004). The DSSC’s efficiency rating ɳ is still approximately half that of the conventional silicon photovoltaics at ~11–14%. There is a major effort, therefore, to develop organic sensitizers that will achieve a more effective harnessing of solar energy.

The outstanding photostability, brightness and FRET-based light channelling properties of GFP-like proteins, provide a real potential for an improved conversion of photons to electricity than the currently used generation of dyes. The spectral diversity and FRET properties of GFP-like proteins covering almost the full visible spectrum is relevant for the strategic undertaking to develop multi-protein arrays to enhance solar collection. We can use a similar design to photosynthetic systems where a large number of light-harvesting antenna pigments channel excitation into a small number of photochemically active pigments in designing GFP-based solar cells. The use of multiple dyes in DSSCs is already being used to increase the absorption of solar cells and increase their efficiency (e.g. Maruyama et al. 1998; Zhang and Jenekhe 2000). Multi-chromophoric GFP arrays, with donor–acceptor FRET pairs, will be suitably arranged to channel excitation into photochemically active layers/centres. The quaternary structure of anthozoan GFPs, which naturally form tetramers, oligomers and higher order protein assemblies, are perfectly suited for the task. The arrays can be used to absorb and re-emit light down-shifted in wavelengths that will be matched to the spectral response of the solar cells.

As certain GFP-like proteins arrays possess electron donor–acceptor capabilities (Bogdanov et al. 2009), these proteins can also form the constituents of the photochemically active components. Owing to their molecular properties, these proteins may exhibit structural integrity and an excellent tertiary folding architecture. The assembly of light harvesting GFPs, targeting parts of the UV and most of the visible solar spectra, together with GFPs and other suitable electroactive proteins that mimic photosynthetic reaction centre protein units, would insure a rapid energy transduction, with a high quantum yield and minimal energy losses. A significant challenge found in solar cells replicating photosynthesis is the ability to achieve long-lived charge separation and prevent charge recombination (Gust et al. 2001). One way to address this challenge has been to integrate successive energy gradients into covalently linked molecular dyads or molecular doublets/dimers that will quickly shuttle electrons away from the excitation site to more stable sites.

Recently, a number of studies confirmed that the electrical energy generation from coral GFPs is an effective strategy. One of the first studies to mimic photosynthesis and use GFPs to generate an electrical current was the development of a molecular-scale biophotodiode consisting of a GFP and viologen (N-allyl-N’-[3-propylamido-N”,N”-di(n-octadecyl)]-4,4’- bipyridium dibromide) as a sensitizer and electron acceptor using Langmuir–Blodgett techniques (Choi et al. 2000).

The same team improved the system by fabricating a bio-photodiode consisting of GFP as the sensitizer and an electron acceptor with a molecular relay – a heterojunction consisting of molecular layers sandwiched between metal electrodes – to achieve faster electron transfer (Choi et al. 2001). This metal/insulator/metal (MIM) structured electronic device was fabricated with heterofilms made of GFP and 10 layers of viologen and TCNQ (N-docosilquinolinium tetracyano dimethan) used as the sensitizer, an electron relay and an electron acceptor, respectively, positioned between the aluminium and indium tin oxide (ITO) glass. Upon light illumination, the GFP electrons were excited from their ground to excited state (GFP*), returned to their ground state with the emission of green fluorescence at 510 nm. Since the electron acceptor was exposed to the excited sensitizer, some of GFP*’s photoexcited electrons were separated (GFP+/viologen/ TCNQ) and transferred (GFP+/viologen/TCNQ) to the TCNQ layer via their redox potential difference, thereby generating a photo-induced electric current (Choi et al. 2001).

Another study used GFP as an electron sensitizer and cytochrome c as an electron acceptor in a self-assembled heterolayer (Choi and Fujihara 2004). The monolayer of thiol-modified cytochrome c was formed on Au-coated glass and GFP was molecularly tethered onto the cytochrome c surface by electrostatic attraction. A relatively efficient photo-induced current was generated with repeated on/off cycles.

One recent study combined two contemporary photovoltaic strategies – the approach of using solid-state semiconductors to produce hole–electron pairs, with the second approach represented by the Grätzel cell (Fig. 7.4) (Chirgwandi et al. 2008). A 30 nm anode–cathode spacing was produced on a silicon chip by electron beam lithography and self-assembled GFP was employed as the dye molecule. The improved lifetime of photo-induced hole–electron pairs in EGFP (enhanced GFP) arrays allowed for the competition of electron–hole recombination with both, the electrons from an electrode and via the de-excitation process, to generate the photo-induced current (Chirgwandi et al. 2008). The electron transport was found to correlate with the excitation cross-section of EGFP, the intensity of incident light, temperature, source–drain potential bias and the gate potential.

Fig 7.4 A 600 μm thick silicon wafer with an insulating SiO2 substrate of 1000 Å thickness, a drop of GFP and aluminium (Al2O3) electrodes on top SiO2 attached to a gold (Au) surface in order to produce an ohmic contact. (a) Schematic cross-section of the device and the measuring system and (b) an overall electron transfer process and current flow, but no current was observed in the absence of the GFP drop (green, represents GFP) (from Chirgwandi et al. 2008).

In a widely publicized, but unpublished study, the same team from Sweden’s Chalmers University of Technology, headed by Z.G. Chirgwandi, generated electricity from GFP acting both as a sensitizer dye and as an electron donor (Knight 2010). A droplet of EGFP assembled itself into strands between a tiny gap of two aluminium electrodes positioned upon a silicon dioxide substrate. When exposed to UV light, EGFP acted as a dyesensitized solar cell and produced electricity. Amazingly, the team even reported to have made the solar cell work in the absence of natural light, using the strategy of bioluminescent light emission from a mixture of chemicals from fireflies (magnesium and luciferase enzymes) to induce fluorescence as a light source for GFP’s capacity to generate electricity. They hypothesized that since GFPs are genetically encodable, this kind of solar cell can be implanted or genetically expressed in the human body to synthesize energy from naturally occurring chemicals.

The predominant degradation of organic solar cells is attributable to the exposure to high-energy photons and the sensitivity of the photoactive layer to oxidative damage. Singlet molecular oxygen and complex photochemical reactions between the different solar cell layers, in combination with moisture, oxygen, light and heat often lead to a degradation and device failure (Brabec et al. 2010). The high photostability of GFPs and their antioxidant properties have the capacity to reduce the potential for photodegradation, thus further increasing their superior properties for photovoltaic technologies.

Fluorescent organic dyes are currently considered to be the most promising luminescent species for building efficient solar cells due to their high quantum efficiencies and absorption coefficients, ease of processing and relatively low cost (Klampaftis et al. 2009). The benefits of GFPs are that they are non-toxic, biodegradable and environmentally friendly. They can be produced cheaply by transfection and growth in bacterial, fungal or plant cultures. Availability of inexpensive and varied raw materials accompanied by an easy fabrication procedure and the ability to tune molecular properties has made GFP-based organic photovoltaics a highly attractive proposition. The truly unique property of GFP-like proteins is that they are genetically encodable and can be transfected into any organism, so that potentially, energy generation can be accomplished within living organisms.

7.3.5 Light-emitting diodes, optoelectronic and photochromic materials

Since the 1980s, both the academic and the private industries have been actively pursuing the development of all-organic materials and devices as alternatives to inorganic-based electronic and optoelectronic systems. The major advantages of organic relative to inorganic technologies are their smaller sizes, simpler fabrication through established lithography techniques, lower cost and large area electronics when coupled with conventional commercial printing technologies. The applications include organic light-emitting devices (OLEDs), lasers, photovoltaic and thin-film transistors.

Today commonly used diodes are semiconductor diodes, either solid or liquid materials, that conduct electricity at room temperature and include chemical elements and compounds such as silicon, germanium, selenium, gallium arsenide, zinc selenide and lead telluride. Light-emitting diodes (LEDs) emit visible light or infrared radiation when an electric current passes through them and are made of semiconductors mixed with substances that absorb electromagnetic radiation and re-emit it as visible light in a process known as electroluminescence. LEDs are employed in flat screen TVs, computer and other device touch screens, numerical displays of electronic digital watches and pocket calculators. An OLED is a flat light-emitting technology, made by placing a series of organic thin films between two conductors that generate bright light when an electrical current is applied. This technology was based on research initiated in the early 1960s (Kallmann and Pope 1960a,Kallmann and Pope 1960b; Peter and Wolfgang 1962). OLEDs are being developed with a hope to replace crystalline/diode semiconductors and produce more efficient and cheaper LEDs.

The first application of GFPs in OLEDs was over a decade ago. A research team at the University of Southern California, headed by M. Thompson, used a biomimetic approach to generate light based on the excitation of GFP in an enzymatic chemiluminescent reaction that occurs in the jellyfish (You et al. 2000). Since the GFP protein scaffold adds an insulating layer around the fluorophore, that may hinder energy or electron transfer between the fluorophore and the surrounding matrix, the team prepared synthetic derivatives of GFP’s imidazolidinone and oxazolone, thus eliminating the scaffold. A stronger electron-donor group was incorporated to increase the electron donating strength. Next, the GFP-like molecules were seeded onto a matrix of organic molecules, which converted energy into light, creating an OLED and eventually produced green and red types (You et al. 2000). Given the enormous variety of GFP-type proteins, many with different fluorophores, carrier transport and other functions, other OLEDs may be prepared, using the insight provided by the jellyfish GFP.

The exciting photoswitching properties of the photoactive fluorescent proteins (PAFPs) described earlier in this chapter can also be employed to develop advanced biomimetic solutions. PAFPs can act as nano-elements that can be switched between spectroscopically different states, enabling optical manipulation of matter and information at nanoscopic scales for optical data storage.

The limitation of the current commercial optical data storage devices (e.g. CD, DVD, Blu-ray discs, etc.) is their two-dimensional structure. To increase the storage data density, optical media using three dimensions (3D) was developed using polymers (Wang and Esener 2000; Gindre et al. 2006). With the development of two-photon excitation (TPE), the manipulation of optically encoded information within a 3D volume (i.e. voxel) became possible, since it enables excitation of molecules in a minute volume of the excitation beam in an opaque material (Mandzhikov et al. 1973).

The use of photochromic organic dyes to design rewritable optical data storage devices was first suggested ~ 50 years ago (Hirschberg 1956) and the science has been rapidly developing during the last decade (e.g. Berkovic et al. 2000; Irie 2000; Yokoyama 2000; Irie et al. 2002). The possibility of using photo-induced cycling between the protonated and the deprotonated forms of wtGFP for data storage at the single molecule level was first suggested by R. Tsien (Tsien 1998). Later, the photoswitching property of PAFPs for 3D data storage media was proposed for single GFP-like protein crystals (PCFPs and RSFPs) in which each protein molecule in the crystal would represent a binary encodable element, depending on whether the protein was in its ‘on’ (bright) or ‘off’ (dark) states (Hell et al. 2007).

PAFPs which have the capacity to alter colours following irradiation by light also provide the basis for data encoding and storage in a single crystal. This capability was recently explored by a multi-national research team from Belgium, Japan, France, USA and Germany (Adam et al. 2010). They pointed out that the advantages of using photoconvertible GFP-like proteins were: (i) that the two different switching/colour modes constituted alternative basis for data encoding and could be combined in a single crystal; (ii) PAFPs enable multicolour data storage; (iii) GFP-like proteins are biodegradable and provide a sustainable solution to the waste recycling of data storage devices. The researchers tested the following proteins: Kaede (Ando et al. 2002), Dronpa (Ando et al. 2004), three mutants of EosFP (Wiedenmann et al. 2004) – the dimeric form d1EosFP, the monomeric form mEosFP (Plate XX) (Wiedenmann et al. 2004), and IrisFP (Fig. 7.3) (Adam et al. 2008). However, they also pointed out several problems due to the biological nature of this technology, such as protein photobleaching, the need for a solvated state, sample aging and thermal instability (Adam et al. 2010). To overcome these problems, systematic screening of marine organisms for novel FPs was suggested, combined with random or rational mutagenesis, in order to generate more photostable and ‘fatigue’ resistant molecules.

The biomimetic exploitation of photoactive GFPs that photoconvert in a reversible or irreversible fashion, and between molecular states that differ in brightness and colour, creates a real possibility of bio-organic data storage devices and computing at nanoscales. Moreover, the photo-induced transformations of GFP-like proteins can be engineered to control the relative orientation and distance of donor–acceptor pairs in organic films. Nanoscale excitation that may be coherent and coupled to protein dipole shifts may generate communicative ‘collective modes’ within protein assemblies and provide a substrate for biological information processing (Hameroff and Schneiker 1987).

The field of photochromic materials in which photoactive GFPs may be used is too vast to be covered here. Light provides a precise and a reversible means of control without the requirement for physical contact. It is suffice to mention that photoresponsive GFP-based films, gels and polymers, with potential other biomimetic functionalities of electron or proton conductivity, of pH-, redox- and ion-responsiveness, are technologically important for a wide range of applications. These may include such devices and technologies as drug delivery and other biochemical and biomedical systems, smart microfluidic devices, microactuators and membranes in which light irradiation and optical control can be applied at micro- or nano-scale resolution and without physical contact. GFP-like protein expression in reef corals can be affected by environmental conditions and biological stresses and in vivo photoactivation of GFP-like proteins can be induced by the build-up of cellular ROS. This biological property can be an important source of biomimetic applications and the creation of photoactive materials that respond to biological and environmental stimuli.

7.4 Conclusion

Nature has produced a dazzling array of outstanding optical and molecular properties of GFPs and a variety of biological functions that have only become apparent in the last decade. This chapter presented several biomimetic applications of GFPs based on these properties and on their functional roles in marine organisms, and in particular, within the biophotonic system made up of the coral–algal symbiotic association.

The use of GFP and of GFP-like proteins has revolutionized the field of cell biology and biomedicine and the importance of the GFP-based technologies has been recognized with the 2008 Nobel Prize in Chemistry awarded to the pioneers in the field – Osamu Shimomura, Martin Chalfie and Roger Y. Tsien. The time has come for the field to evolve further so that biomimetic methods for improved energy production, and of biophotonic and optoelectronic developments emerge based on GFP’s unique optical, molecular and biological properties.

Furthermore, GFP-like proteins are no longer considered to be inert when expressed in cells and may have a variety of functions linked to their photoactivity and to photon, electron and proton channelling properties. It may soon be possible to use GFPs not only as imaging tools or bio-sensors but as actuators with capacity to control a variety of biological functions.

During the last decade, the emergence of the use of optical methods in combination with genetic manipulations and light-driven control of cellular activity have created a new field known as ‘optogenetics’ (Miesenböck 2009). The ability to express GFPs in specific cellular organelles or compartments and fused to proteins of interest has become the norm. The ability to induce light-driven redox, membrane potential or electrolytic changes may provide an exciting possibility to genetically encode a light-inducible biological function and to precisely control it, using GFPs as optogenetic actuators. By further understanding the biology of GFPs in marine organisms, we can learn to use them as the basis for many new applications. It may even be feasible to develop GFP-based genetically encodable, self-powered nanodevices or even biological computers that record and transmit information, with wide-ranging applications in medicine, biological sciences and environmental monitoring.

7.5 References

Ackleson, S. G. Light in shallow waters: a brief research review. Limnol Oceanogr. 2003; 48:323–328.

Adam, V., Lelimousin, M., Boehme, S., Desfonds, G., Nienhaus, K., Field, M. J., Wiedenmann, J., McSweeney, S., Nienhaus, G. U., Bourgeois, D. Structural characterization of IrisFP, an optical highlighter undergoing multiple photoinduced transformations. Proc Natl Acad Sci USA. 2008; 105:18343–18348.

Adam, V., Mizuno, H. A., Grichine, A., Hotta, J. -I., Yamagata, Y., Moeyaert, B., Nienhaus, G. U., Miyawaki, A., Bourgeois, D., Hofkens, J. Data storage based on photochromic and photoconvertible fluorescent proteins. J Biotechnology. 2010; 149(4):289–298.

Aglyamova, G. F., Hunt, H. E., Chintan, K., Modib, M., Matz, M. V. Multicolored homologs of the green fluorescent protein from hydromedusa Obelia sp. Photochem & Photobiol Sci. 2011; 10:1303–1309.

Agmon, N. Proton pathways in green fluorescence protein. Biophys J. 2005; 88:2452–2461.

Alieva, N. O., Konzen, K. A., Field, S. F., Meleshkevitch, E. A., Beltran-Ramirez, V., Miller, D. J., Salih, A., Wiedenmann, J., Matz, M. V. Diversity and evolution of coral fluorescent proteins. PLoS One. 2008; 3:e2680.

Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. An optical marker based on the UV-induced green-to-red-photoconversion of a fluorescent protein. Proc Natl Acad Sci USA. 2002; 99:12651–12656.

Andresen, M., Wahl, M. C., Stiel, A. C., Grater, F., Schafer, L. V., Trowitzsch, S., Weber, G., Eggeling, C., Grubmuller, H., Hell, S. W., et al. Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proc Natl Acad Sci USA. 2005; 102:13070–13074.

Asada, K. Update on reactive oxygen species in chloroplasts: production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiology. 2006; 141:391–396.

Baird, G. S., Zacharias, D. A., Tsien, R. Y. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA. 2000; 2000(97):11984–11989.

Bastiaens, P. I. H., Pepperkok, R. Observing proteins in their natural habitat: the living cell. Trends in Biological Science. 2000; 25:631–637.

Berkovic, G., Krongauz, V., Weiss, V. Spiropyrans and spirooxazines for memories and switches. Chem Rev. 2000; 100:1741–1753.

Bogdanov, A. M., Mishin, A. S., Yampolsky, I. V., Belousov, V. V., Chudakov, D. M., Subach, F. V., Verkhusha, V. V., Lukyanov, S., Lukyanov, K. A. Green fluorescent proteins are light-induced electron donors. Nat Chem Biol, 26. 2009; 5(7):459–461.

Bomati, E. K., Manning, G., Deheyn, D. D. Amphioxus encodes the largest known family of green fluorescent proteins, which have diversified into distinct functional classes. BMC Evol Biol. 2009; 9:77.

Bou–Abdallah, F., Chasteen, N. D., Lesser, M. P. Quenching of superoxide radicals by green fluorescent protein. Biochim Biophys Acta, Gen Subj. 2006; 1760:1690–1695.

Brabec, C. J., Gowrisanker, S., Halls, J. J. M., Laird, D., Jia, S., Williams, S. P. Polymer–fullerene bulk-heterojunction solar cells. Adv Mater. 2010; 22:3839–3856.

Brown, B. E. Coral bleaching: causes and consequences. Coral Reefs. 1997; 16(Supplem):S129–S138.

Catala, R. Fluorescence effect from corals irradiated with ultra-violet rays. Nature. 1959; 1959(183):949.

Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science. 1994; 1994(263):802–805.

Chalfie, M. Green fluorescent protein. Photochem Photobiol. 1995; 62:651–656.

Chalfie, M., Kain, S., Green Fluorescent Protein: Properties, Applications, and Protocols. 2. Wiley-Interscience. Hoboken, NY, 2006.

Chattoraj, M., King, B. A., Bublitz, G. U., Boxer, S. G. Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc Natl Acad Sci. 1996; 93:8362–8367.

Chirgwandi, Z. G., Panas, I., Johansson, L. -G., Norden, B., Willander, M., Winkler, D., Ågren, H. Properties of a biophotovoltaic nanodevice. J Phys Chem Lett. 2008; 112:18717–18721.

Chishima, T., Miyagi, Y., Wang, X., Yamaoka, H., Shimada, H., Moossa, A. R., Hoffman, R. M. Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression. Cancer Res. 1997; 57:2042–2047.

Choi, J. W., Nam, Y. S., Oh, B. K., Lee, W. H., Fujihira, M. Artificial molecular photodiode consisting of GFP/viologen hetero-thin film. Synth Met. 2000; 117:241.

Choi, J. W., Nam, Y. S., Lee, W. H. Rectified photocurrent of the protein-based bio-photodiode. Appl Phys Lett. 2001; 79(10):570–572.

Choi, J. -W., Fujihira, M. Molecular-scale biophotodiode consisting of a green fluorescent protein assembled heterolayer. Appl Phys Lett. 2004; 84:2187–2189.

Chuang, W. T., Chen, B. S., Chen, K. -Y., Hsieh, C. -C., Chou, P. T. Fluorescent protein red Kaede chromophore; one-step, high-yield synthesis and potential application for solar cells. Chem Commun. 2009; 2009:6982–6984.

Chudakov, D. M., Verkhusha, V. V., Staroverov, D. B., Souslova, E. A., Lukyanov, S., Lukyanov, K. A. Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol, 22(11), 1435–9. Epub 2004. (Oct 24):2004.

Chudakov, D. M., Matz, M. V., Lukyanov, S. A., Lukyanov, K. A. Fluorescent proteins and their applications to imaging in living cells and tissues. Physiol Rev. 2010; 2010(90):1103–1163.

Chudakov, D. M., Belousov, V. V., Zaraisky, A. G., Novoselov, V. V., Staroverov, D. B., Zorov, D. B., Lukyanov, S., Lukyanov, K. A. Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol. 2003; 2003(21):191–194.

Cody, C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G., Ward, W. W. Chemical structure of the hexapeptide chromophore of the Aequorea green- fluorescent protein. Biochemistry. 1993; 32:1212–1218.

Cox, G., Salih, A. Fluorescent lifetime imaging of symbionts and fluorescent proteins in reef corals. Multiphoton Microscopy in the Biomedical Sciences V. San Jose, USA: Proc SPIE; 2005.

Cox, G., Salih, A. Fluorescent characteristics of fluoro-chromatophores in corals. Focus on Multidimensional Microscopy. Cheng P C, Hwang P P, Wu J L, Wang G and Kim H (eds). World Scientific Publishing Co. 2006; 3:145–151.

Cox, G., Matz, M., Salih, A. Fluorescence lifetime imaging of coral fluorescent proteins. Microscopy Res Technique. 2007; 70:243–251.

Day, R. N., Davidson, M. W. The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev. 2009; 2009(38):2887–2921.

Deisenhofer, J., Epp, O., Miki, K., Huber, R., Michel, H. X-ray structure analysis of a membrane protein complex. Nature. 1985; 318:618.

Dove, S., Takabayashi, M., Hoegh-Guldberg, O. Isolation and partial characterisation of the pink and blue pigments of pocilloporid and acroporid corals. Biol Bull Mar Biol Lab, Woods Hole. 1995; 189:288–297.

Dove, S. G., Hoegh-Guldberg, O., Ranganathan, S. Major colour patterns of reef-building corals are due to a family of GFP-like proteins. Coral Reefs. 2001; 19:197–204.

Dove, S. G., Lovell, C., Fine, M., Deckenback, J., Hoegh-Guldberg, O., Iglesias-Prieto, R., Anthony, K. R. N. Host pigments: potential facilitators of photosynthesis in coral symbioses. Plant Cell Environ. 2008; 31:1523–1533.

Eaton, W. A., Hofrichter, J. Sickle cell hemoglobin polymerization. Adv Protein Chem. 1990; 40:63–79.

Evdokimov, A. G., Pokross, M. E., Egorov, N. S., Zaraisky, A. G., Yampolsky, I. V., Merzlyak, E. M., Shkoporov, A. N., Sander, I., Lukyanov, K. A., Chudakov, D. M. Structural basis for the fast maturation of Anthropoda green fluorescent protein. EMBO Rep. 2006; 7:1006–1012.

Field, S. F., Bulina, M. Y., Kelmanson, I. V., Bielawski, J. P., Matz, M. V. Adaptive evolution of multicolored fluorescent proteins in red building corals. J Mol Evol. 2006; 62:332–339.

Förster, T. Intermolecular energy migration and fluorescence. Ann Phys. 1948; 2:55–75.

Förster, T. In Modern Quantum Chemistry. Istanbul Lectures, O. Sinanoglu Academic, New York. 1965; 3:93–137.

Franklin, K. A., Whitelam, G. C. Phytochrome a function in red light sensing. Plant Signal Behav, Sep–Oct;. 2007; 2(5):383–385.

Gaffron, H., Wohl, K. Zur Theorie der Assimilation. Naturwiss. 1936; 24:81–90.

Gilmore, A. M., Larkum, A. W. D., Salih, A., Itoh, S., Shibata, Y., Bena, C., Yamasaki, H., Papina, M., Van Woesik, R. Simultaneous time-resolution of the emission spectra of fluorescent proteins and zooxanthellae chlorophyll in reef-building corals. Photochem Photobiol. 2003; 77:515–523.

Gindre, D., Boeglin, A., Fort, A., Mager, L., Dorkenoo, K. D. Rewritable optical data storage in azobenzene copolymers. Opt Expr. 2006; 14:9896–9901.

Grant, D. M., Zhang, W., McGhee, E. J., Bunney, T. D., Talbot, C. B., Kumar, S., Munro, I., Dunsby, C., Neil, M. A. A., Katan, M., French, P. M. W. Multiplexed FRET to image multiple signaling events in live cells. Biophys J. 2008; 95:L69–L71.

Green, B. R. The evolution of light harvesting antennas. In: Green B. R., Parson W. W., eds. Light Harvesting Antennas in Photosynthesis. Dordrecht: Kluwer Academic Press; 2003:129–168.

Gregg, B. A., Hanna, M. C. Comparing organic to inorganic photovoltaic cells: Theory, experiment and simulation. J Appl Phys. 2003; 2003(93):3605.

Gross, L. A., Baird, G. S., Hoffman, R. C., Baldridge, K. K., Tsien, R. Y. The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA. 2000; 97:11,990–11,995.

Gruber, D. F., Kao, H. -T., Janoschka, S., Tsai, J., Pieribone, V. Patterns of fluorescent protein expression in scleractinian corals. Biolog Bull. 2008; 215(2):143–154.

Gust, D., Moore, T. A., Moore, A. L. Mimicking photosynthetic solar energy transduction. Acc Chem Res. 2001; 34:40–48.

Habuchi, S., Ando, R., Dedecker, P., Verheijen, W., Mizuno, H., Miyawaki, A., Hofkens, J. Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc Natl Acad Sci USA. 2005; 102:9511–9516.

Hameroff, S., Schneiker, C. Ultimate Computing: Biomolecular Consciousness and Nanotechnology. Elsevier-North Holland. 1987.

Hardin, B. E., Hoke, E. T., Armstrong, P. B., Yum, J. -H., Comte, P., Torres, T., Fréchet, J. M. J., Nazeeruddin, M. K., Grätzel, M., McGehee, M. D. Increased light harvesting in dye-sensitized solar cells with energy relay dyes. Nature Photonics. 2009; 3:406–411.

Heim, R., Prasher, D. C., Tsien, R. Y. Wavelength mutations and post translational autoxidation of green fluorescent protein. Proc Natl Acad Sci. 1994; 91:12501–12504.

Hell, S. W., Jakobs, S., Andresen, M., Stiel, A. C., Eggeling, C., Method and apparatus for storing a three-dimensional arrangement of data bits in a solid-state body, 2007. [Patents #US20070047287, DE102005040671, EP1763024].

Hirschberg, Y. Reversible formation and eradication of colors by irradiation at low temperatures: a photochemical memory model. J Am Chem Soc. 1956; 78:2304–2312.

Hoegh-Guldberg, O. Climate change, coral bleaching and the future of the world’s coral reefs. Mar Freshw Res. 1999; 50:839–866.

Hoffman, R. M. The multiple uses of fluorescent proteins to visualize cancer in vivo. Nature Rev Cancer. 2005; 5:796–806.

Hollingsworth, L. L., Kinzie, R. A., Lewis, T. D., Krupp, D. A., Leong, J. A. C. Phototaxis of motile zooxanthellae to green light may facilitate symbiont capture by coral larvae. Coral Reefs. 2005; 24:523.

Hopf, M., Gohring, W., Ries, A., Timpl, R., Hohenester, E. Crystal structure and mutational analysis of a perlecan-binding fragment of nidogen-1. Nat Struct Biol. 2001; 8:634–640.

Hoppe, H., Sariciftci, N. S. Organic solar cells: an overview. Mater Res. 2004; 19(7):1924–1945.

Hunt, M. E., Scherrer, M. P., Ferrari, F. D., Matz, M. V. Very bright green fluorescent proteins from the Pontellid copepod Pontella mimocerami. PLoS One. 2010; 5:e11517.

Imamoto, Y., Kataoka, M. Structure and photoreaction of photoactive yellow protein, a structural prototype of the PAS domain superfamily. Photochemi Photobiol. 2007; 83(1):40–49.

Irie, M. Diarylethenes for memories and switches. Chem Rev. 2000; 100:1685–1716.

Irie, M., Fukaminato, T., Sasaki, T., Tamai, N., Kawai, T. Organic chemistry: a digital fluorescent molecular photoswitch. Nature. 2002; 420:759–760.

Jain, V., Rajbongshi, B. K., Mallajosyula, A. T., Bhattacharjya, G., KumarIyer, S. S., Ramanathan, G. Photovoltaic effect in single-layer organic solar cell devices fabricated with two new imidazolin-5-one molecules. Solar Energy Mater Solar Cells. 2008; 92:1043–1046.

Jerlov, N. G. Marine optics. Oxford: Elsevier Scientific Publishing Company; 1976.

Kahng, S. E., Salih, A. Localization of fluorescent pigments in a non- bioluminescent, azooxanthellate octocoral suggests a photo-protective function. Coral Reefs. 2005; 24:436–437.

Kallmann, H., Pope, M. Positive hole injection into organic crystals. J Chemi Physi. 1960; 32:300.

Kallmann, H., Pope, M. Bulk conductivity in organic crystals. Nature. 1960; 186(4718):31.

Kao, H. T., Sturgis, S., DeSalle, R., Tsai, J., Davis, D., Gruber, D. F., Pieribone, V. A. Dynamic regulation of fluorescent proteins from a single species of coral. Marine Biotechnol. 2007; 1:1–14.

Karvanos, G. J. Fundamentals of Photoinduced Electron Transfer. New York: VCH; 1993.

Kawaguti, S. On the physiology of reef corals. VI. Study, of the pigments. Palao Trop Biol Stn Stud. 1944; 2:617–674.

Kelmanson, I. V., Matz, M. Molecular basis and evolutionary origins of color diversity in great star coral Montastraea cavernosa (Scleractinia: Faviida). Mol Biol Evol. 2003; 20:1125–1133.

Klampaftis, E., Ross, D., McIntosh, K. R., Richards, B. S. Enhancing the performance of solar cells via luminescent down-shifting of the incident spectrum: A review. Solar Energy Materi Solar Cells. 2009; 93:1182–1194.

Knight, H. Green machine: Squeezing solar juice from jellyfish. New Scientist http://www. newscientist. com/article/dn19416, 2010. [green-machine-squeezing- solar-juice-from-jellyfish. html].

Kulzer, F., Orrit, M. Single–molecule optics. Ann Rev Phys Chem. 2004; 55:585–611.

Labas, Y. A., Gurskaya, N. G., Yanushevich, Y. G., Fradkov, A. F., Lukyanov, K. A., Lukyanov, S. A., Matz, M. V. Diversity and evolution of the green fluorescent protein family. Proc Natl Acad Sci USA. 2002; 99:4256–4261.

Lesser, M. P. Coral reefs bleaching and global climate change: Can corals survive the next generation? Proc Natl Acad Sci. 2007; 104:5259–5260.

Li, I. T., Pham, E., Truong, K. Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics. Biotechnol Lett. 2006; 28:1971–1982.

Lippincott–Schwartz, J., Snapp, E., Kenworthy, A. Studying protein dynamics in living cells. Nature Rev Mol Cell Biol. 2001; 2:444–456.

Lippincott-Schwarz, J. Cell biology: ripping up the nuclear envelope. Nature. 2002; 416(6876):31–32.

Lippincott-Schwartz, J., Patterson, G. H. Fluorescent proteins for photo-activation experiments. Methods Cell Biology. 2008; 85:45–61.

Liu, H. R., Ramnarayanan, R., Logan, E. B. Production of electricity during wastewater treatment using a single chamber microbial fuel cell. Environ Sci Technol. 2004; 38(7):2281–2285.

Losi, A. The bacterial counterparts of plant phototropins. Photochem, Photobiol Sci. 2004; 3:566–574.

Lukyanov, K. A., Fradkov, A. F., Gurskaya, N. G., Matz, M. V., Labas, Y. A., Savitsky, A. P., Markelov, M. L., Zaraisky, A. G., Zhao, X., Fang, Y., Tan, W., Lukyanov, S. A. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J Biol Chem. 2000; 275:25879–25882.

Lukyanov, K. A., Chudakov, D. M., Lukyanov, S., Verkhusha, V. V. Innovation: photoactivatable fluorescent proteins. Nat Rev Mol Cell Biol. 2005; 6(11):885–891.

Lukyanov, K. A., Loschenov, V. B., Luker, G. D., Chudakov, D. M. Near- infrared fluorescent proteins. Nat Methods. 2010; 7(10):827–829.

Mandzhikov, V. F., Murin, V. A., Barachevskii, V. A. Nonlinear coloration of photochromic spiropyran solutions. Sov J Quant Electron. 1973; 3:128–129.

Maruyama, T., Shinyashiki, Y., Osako, S. Energy conversion efficiency of solar cells coated with fluorescent coloring agent. Solar Energy Mater Solar Cells. 1998; 56:1–6.

Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L., Lukyanov, S. A. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol. 1999; 17:969–973.

Matz, M. V., Labas, Y. A., Ugalde, J. Evolution of function and color in GFP- like proteins. Methods Biochem Anal. 2006; 47:139–161.

Mazel, C. H. Spectral measurements of fluorescence emission in Caribbean cnidarians. Mar Ecol Prog Ser. 1995; 120:185–191.

Mazel, C. H., Lesser, M. P., Gorbunov, M. Y., Barry, Y. M., Farrell, J. H., Wyman, K. D., Falkowski, P. G. Green fluorescent proteins in Caribbean corals. Limnol Oceanogr. 2003; 48:402–411.

Mank, M., Griesbeck, O. Genetically encoded calcium indicators. Chem Rev. 2008; 108:1550–1564.

Meech, S. R. Proton transfer – excited state reactions in fluorescent protein. Chem Soc Rev. 2009; 38:2922–2934.

Miesenböck, G. The optogenetic catechism. Rev Sci. 2009; 326:395–399.

Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., Tsien, R. Y. Fluorescent indicators for Ca2 + based on green fluorescent proteins and calmodulin. Nature. 1997; 388:882–887.

Miyawaki, A., Tsien, R. Y. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods in Enzymology. 2000; 327:472–500.

Miyawaki, A., Mizuno, H., Nagal, T., Sawano, A. Development of genetically encoded fluorescent indicators for calcium. Methods in Enzymology. 2003; 360:202–225.

Miyawaki, A. Innovations in the imaging of brain functions using fluorescent proteins. Neuron. 2005; 48:189–199.

Miyawaki, A. Great expectations. Science. 2009; 326(5951):339.

Mizuno, H., Mal, T. K., Tong, K. I., Ando, R., Furuta, T., Ikura, M., Miyawaki, A. Photo–induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Mol Cell. 2003; 12:1051–1058.

Morin, J. G., Hastings, J. W. Energy transfer in a bioluminescent system. J Cell Physiol. 1971; 1971(77):313.

Morise, H., Shimomura, O., Johnson, F. H., Winant, J. Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry. 1974; 13(12):2656–2662.

Muscatine, L., Falkowski, P., Porter, J., Dijbinsky, Z. Fate of photosynthetically-fixed carbon in light and shade adapted colonies of the symbiotic coral Stylophora pistillata. Proc R Sot Lond Ser B. 1984; 222:181–202.

Ong, W. J., Alvarez, S., Leroux, I. E., Shahid, R. S., Samma, A. A., Peshkepija, P., Morgan, A. L., Mulcahy, S., Zimmer, M. Function and structure of GFP-like proteins in the protein data bank. Mol Biosyst. 2011; 7(4):984–992.

Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., Remington, S. J. Crystal structure of the Aequorea victoria green fluorescent protein. Science. 1996; 273:1392–1395.

Oswald, F., Schmitt, F., Leutenegger, A., Ivanchenko, S., D’Angelo, C., Salih, A., Maslakova, S., Bulina, M., Schirmbeck, R., Nienhaus, G. U., Matz, M. V., Wiedenmann, J. Contributions of host and symbiont pigments to the coloration of reef corals. FEBS J. 2007; 274:1102–1109.

Palmer, C. V., Modi, C. K., Mydlarz, L. D. Coral fluorescent proteins as antioxidants. PLoS ONE. 2009; 4(10):e7298.

Patterson, G. H., Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science. 2002; 297:1873–1877.

Patterson, G. H., Fluorescent proteins for cell biology vol. 411Reporter Genes, D., Practical Guide, A., Anson. From: Methods in Molecular Biology. © Humana Press Inc, Totowa, NJ, 2007:47–80.

Peter, M., Wolfgang, H. Space-charge-limited currents in organic crystals. J Appli Phys. 1962; 33:205.

Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., Cormier, M. J. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992; 111:229–233.

Prescott, M., Salih, A., Chapter 4: Genetically encoded fluorescent probes and their applications in the life sciencesGoldys, E., eds.. Fluorescence Applications in Biotechnology and Life Sciences. John Wiley & Sons, Inc, Hoboken, New Jersey. USA & Canada, 2009:55–77.

Quillin, M. L., Anstrom, D. M., Shu, X., O’Leary, S., Kallio, K., Lukyanov, K. A., Remington, S. J. The kindling fluorescent protein from Anemonia sulcata: dark state structure at 1. 38Å resolution. Biochemistry. 2005; 44:5774–5787.

Remington, S. J., Hanson, G. T., Cannon, M., Aggeler, R., Oglesbee, D., Capaldi, R. A., Tsien, R. Y. Third generation GFP biosensors for real time readout of pH and redox potential in living cells. Biomed Opt. 2004; 5329:1–12.

Remington, G. Fluorescent proteins: maturation, photochemistry and photo-physics. Current Opinion Struct Biol. 2006; 16:714–721.

Rosenow, M. A., Huffman, H. A., Phail, M. E., Wachter, R. M. The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization-oxidation-dehydration mechanism for chromophore maturation. Biochemistry. 2004; 43:4464–4472.

Roth, M. S., Latz, M. I., Goericke, R., Deheyn, D. D. Green fluorescent protein regulation in the coral Acropora yongei during photoacclimation. J Exp Biol. 2010; 213:3644–3655.

Salih, A., Hoegh-Guldberg, O., Cox, G. Photoprotection of symbiotic dinoflagellates by fluorescent pigments in reef corals, Proc Aust Coral Reef Society 75 Anniv Conference. Greenwood & Hall, pp. 217–230, 1998.

Salih, A., Larkum, A. W. D., Cox, G., Kühl, M., Hoegh-Guldberg, O. Fluorescent pigments in corals are photoprotective. Nature. 2000; 408:850–853.

Salih, A., Larkum, A. W. D., Cox, G. Photoprotection from photoinhibion of symbiotic algae in corals by fluorescent pigments. PS2001 Proceedings: 12th International Congress on Photosynthesis, CSIRO Publish: Melbourne, Australia. http://www. publish. csiro. au/ps2001/cf/search/article. cfm?ID=4530661, 2001.

Salih, A., Cox, G., Larkum, A. W. D. Cellular organization and spectral diversity of GFP-like proteins in coral cells studied by single and multi-photon imaging and microspectroscopy. Multiphoton microscopy in Biomedical Sciences III, (Pereasamy A and So P T (eds)). Proceedings of SPIE, California. 2003; 4963:194–200.

Salih, A., Cox, G., Szymczak, R., Coles, S., Baird, A., Dunstan, A., Cocco, G., Mills, J., Larkum, A. W. D. The role of host-based color and fluorescent pigments in photoprotection and in reducing bleaching stress in corals. In Proceedings of the 10th International Coral Reef Symposium. Okinawa, Japan: ICRS. http://www. coralcoe. org. au/pub-scientific2006. htm, 2006.

Salih, A., Wiedenmann, J., Mikhail, M., Larkum, A. W. D., Cox, G. Photoinduced activation of GFP-like proteins in tissues of reef corals. Proc SPIE. 6098, 2006.

Schlichter, D., Fricke, H. W. Coral host improves photosynthesis of endosymbiotic algae. Naturwissenschaften. 1990; 77:447–450.

Shagin, D. A., et al. GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol Biol Evol. 2004; 21:841–850.

Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E., Tsien, R. Y. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol. 2004; 22:1567–1572.

Shaner, N. C., Patterson, G. H., Davidson, M. W. Advances in fluorescent protein technology. J Cell Science. 2007; 120(24):4247–4259.

Shcherbo D, Shemiakina I I, Ryabova A V, Luker K E, Schmidt B T, Souslova E A, Gorodnicheva T V, Strukova L, Shidlovskiy K M, Britanova O V and Zaraisky A G, Fluorescent indicators for Ca2 + based on green fluorescent proteins and calmodulin.

Shcherbo, D., Merzlyak, E. M., Chepurnykh, T. V., Fradkov, A. F., Ermakova, G. V., Solovieva, E. A., Lukyanov, K. A., Bogdanova, E. A., Zaraisky, A. G., Lukyanov, S., Chudakov, D. M. Bright farred fluorescent protein for whole-body imaging. Nat Methods. 2007; 4(9):741–746.

Shimomura, O., Inoue, S., Johnson, F. H., Haneda, Y. Widespread occurrence of coelenterazine in marine bioluminescence. Comp Biochem Physiol, Part B: Biochem Mol Biol. 1980; 65:435–437.

Shimomura, O. The discovery of aequorin and green fluorescent protein. J Microscopy. 2005; 217(1):3–15.

Shinobu, A., Agmon, N. Mapping proton wires in proteins: carbonic anhydrase and GFP chromophore biosynthesis. J Phys Chem A. 2009; 113:7253–7266.

Stepanenko, O. V., Shcherbakova, D. M., Kuznetsova, I. M., Turoverov, K. K., Verkhusha, V. V. Modern fluorescent proteins: from chromophore formation to novel intracellular applications. BioTechniques. 2011; 51:313–327.

Stepanenko, V., Verkhusha, V. V., Kuznetsova, I., Uversky, V. N. Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes. Current Protein Peptide Sci. 2008; 9:338–369.

Stoner-Ma, D., Jaye, A. A., Matousek, P., Towrie, M., Meech, S. R., Tonge, P. J. Observation of excited-state proton transfer in green fluorescent protein using ultrafast vibrational spectroscopy. J Am Chem Soc. 2005; 127:2864–2865.

Takabayashi, M., Hoegh-Guldberg, O. Ecological and physiological differences between two colour morphs of the coral Pocillopora damicornis. Mar Biol. 1995; 123:705–714.

Tsien, R. Y. Green fluorescent protein: review. Ann Rev Biochem. 1998; 67:509–544.

Tsien, R. Y. Nobel lecture: constructing and exploiting the fluorescent protein paintbox. Integr Biol. 2010; 2:77–93.

Tsutsui, H., Karasawa, S., Okamura, Y., Miyawaki, A. Improving membrane voltage measurements using FRET with new fluorescent proteins. Nature Methods. 2008; 5:683–685.

van der Horst, M. A., Hellingwerf, K. J. Photoreceptor proteins, ‘star actors of modern times’: a review of the functional dynamics in the structure of representative members of six different photoreceptor families. Acc Chem Res. 2004; 37:13–20.

van Thor, J. J., Georgiev, G. Y., Towrie, M., Sage, J. T. J. Ultrafast and low barrier motions in the photoreactions of the green fluorescent protein. Biol Chem. 2005; 280:33652–33659.

Veal, C. J., Carmi, M., Dishon, G., Sharon, Y., Michael, K., Tchernov, D., Hoegh-Guldberg, O., Fine, M. Shallow water wave lensing in coral reefs: a physical and biological case study. J Expe Biol. 2010; 213:4304–4312.

Verkhusha, V. V., Lukyanov, K. A. The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat Biotechnol. 2004; 22:289–296.

Wachter, R. M., Remington, S. J. Sensitivity of the yellow variant of green fluorescent protein to halides and nitrate. Curr Biol. 1999; 9:R628–R629.

Wachter, R. M., Watkins, J. L., Hanseong, K. Mechanistic diversity of red fluorescence acquisition by GFP-like proteins. Biochem. 2010; 49:7417–7427.

Wall, M. A., Socolich, M., Ranganathan, R. The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat Struct Biol. 2000; 7:1133–1138.

Wang, M. M., Esener, S. C. Three-dimensional optical data storage in a fluorescent dye-doped photopolymer. Appl Opt. 2000; 39:1826–1834.

Weiss, V. M. Cellular mechanisms of cndiarian bleaching: stress causes the collapse of symbiosis. J Exp Biol. 2008; 211:3059–3066.

Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K., Nienhaus, G. U. EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci USA. 2004; 101:15905–15910.

Wu, B., Piatkevich, K. D., Lionnet, T., Singer, R. H., Verkhusha, V. V. Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics. Curr Opin Cell Biol. 2011; 23:310–317.

Yang, F., Moss, L. G., Phillips, G. N., Jr. The molecular structure of green fluorescent protein. Nat Biotechnol. 1996; 14:1246–1251.

Yanushevich, Y. G., Staroverov, D. B., Savitsky, A. P., Fradkov, A. F., Gurskaya, N. G., Bulina, M. E., Lukyanov, K. A., Lukyanov, S. A. A strategy for the generation of non-aggregating mutants of Anthozoa fluorescent proteins. FEBS Letts. 2002; 511(1–3):11–14.

Yarbrough, D., Wachter, R. M., Kallio, K., Matz, M., Remington, S. J. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2. 0-Å resolution. Proc Natl Acad Sci USA. 2001; 98:462–467.

Yokoyama, Y. Fulgides for memories and switches. Chem Rev. 2000; 100:1717–1739.

You, Y., He, Y., Burrows, P. E., Forrest, S. R., Petasis, N. A., Thompson, M. E. Fluorophores related to the green fluorescent protein and their use in optoelectronic devices. Advanced Materials. 2000; 12(22):1678–1681.

Zhang, X., Jenekhe, S. A. Electroluminescence of multicomponent conjugated polymers. 1. Roles of polymer/polymer interfaces in emission enhancement and voltage-tunable multicolor emission in semiconducting polymer/polymer heterojunctions, Macromolecules. 2000; 33(6):2069–2082.

Zhang, L., Patel, H. N., Lappe, J. W., Wachter, R. M. Reaction progress of chromophore biogenesis in green fluorescent protein. J Am Chem Soc. 2006; 128:4766–4772.