Chapter 2: Optical applications of biomolecules – Optical Biomimetics

2

Optical applications of biomolecules

M.J. Ranaghan, N.L. Wagner, M.N. Sandberg and R.R. Birge,     University of Connecticut, USA

Abstract:

From kinematics to molecular machines, biologically inspired technologies harness and enhance the intrinsic properties of naturally occurring materials and systems for applied technologies. Bacteriorhodopsin (BR) represents the most studied protein for photonic applications, and has found use in artificial retinas, associative and volumetric memories, optical limiters, photovoltaic cells and other devices. The native BR protein is rarely optimal for device applications, and genetic engineering plays an important role in the optimization process. In addition, new retinal proteins such as proteorhodopsin and channelrhodopsin-2 have been discovered which provide new options and opportunities.

Key words

bacteriorhodopsin

cyclicity

biophotonic devices

retinal analogs

genetic engineering

2.1 Introduction: biomimetics and biotechnology

The natural world has long inspired the work of artists and inventors. From kinematics to molecular machines, biologically inspired technologies harness and enhance the intrinsic properties of naturally occurring materials and systems for applied technologies (Tian et al., 2006; Large et al., 2007; Di Natale et al., 2008; Gorb, 2008; Cutkosky and Kim, 2009; Tamerler and Sarikaya, 2009). Natural selection, in particular, has optimized each of these molecules for a specific function in a highly competitive environment. Hence, the evolution of these systems has provided an excellent model for numerous technologies, most of which have yet to be realized on a commercial level.

Molecular electronics is a field that encompasses the storage and retrieval of information at the molecular level. Because these systems can be created from the ‘bottom-up,’ these technologies are believed to be the next phase of data storage and manipulation once lithographic technologies become size limited. This changeover is predicted to occur within the next two decades based on extrapolation of Moore’s Law (Birge, 1994b).

Designs for both organic and inorganic biomimetic devices exist, but bacteriorhodopsin (BR) is often the exemplary biophotonic material for a vast array of applications (see section 2.3 for a comprehensive list of BR-based devices proposed to date). Soviet scientists first explored the unique photophysical properties of BR as a real-time holographic medium, which was termed Biochrome (Hong, 1994; Birge et al., 1999). Many of the details related to the materials produced during this time are vague, because they are classified by the Russian military, but a historical account of this period is available (Vsevolodov, 1998). This reference is also the most comprehensive description of the history, application, and optimization of BR and is an excellent introduction into the field of biomolecular electronic devices.

2.2 Retinylidene proteins for optical devices

Retinylidene proteins are a family of photoactive, integral transmembrane proteins that are found in all three domains of life (Béjà et al., 2000; Spudich et al., 2000; Béjà et al., 2001; Terakita, 2005). These proteins encompass a vast array of functions that include vision (Goldsmith, 1994; Yoshizawa, 1994; Shichida and Imai, 1998; Nathans, 1999), light-transduction (Birge, 1981; Keszthelyi, 1988; Sineshchekov and Spudich, 2004), ion translocation (Lanyi, 1986; Nagel et al., 2003), and photo taxis (Spudich et al., 1989; Takahashi et al., 1990; Spudich et al., 1997; Ren et al., 2001) among others (Max et al., 1995; Max et al., 1998; Provencio et al., 1998; Terakita, 2005). Despite their function, all retinylidene proteins contain an organic chromophore (i.e. vitamin A aldehyde or retinal) that is covalently bound to a conserved lysine residue in helix 7 (Luecke et al., 1999). Upon the absorption of a photon, the retinal chromophore isomerizes and induces a change in the protein structure. This excited state then undergoes a series of thermally driven conformational changes as the protein relaxes back to the resting state.

This family of proteins are divided into two main classes based on their specific role: (1) microbial ion-channel pumps and sensors, and (2) G-protein coupled receptors (Terakita, 2005; Sharma et al., 2007). The type I proteins, which are often represented by BR, have the ability to photocycle and are capable of many such events without regeneration of the photoactive element (Oesterhelt et al., 1991; Hirayama et al., 1992; Sasaki and Spudich, 1999). Most of the type II proteins undergo a photobleaching process that results in an inactive form of the protein. These proteins are often represented by bovine rhodopsin and are not considered useful for devices. Hence, it is the photocyclic mechanism of the type I proteins, specifically BR, that makes the protein a useful biomaterial. Application of this photocyclic mechanism is discussed further in section 2.3.

2.2.1 The bacteriorhodopsin photocycle

The photocycle of BR represents a series of transient photochemical states experienced by the protein after the absorption of a photon. Figure 2.1 shows the currently accepted progression of photostates through the BR photocycle (Lanyi, 2000; Gillespie et al., 2002). Each state is represented by a spectrally discrete conformation of the protein as a proton is transported through the molecule and across the membrane. This process is well characterized, after almost four decades of research, and several models have been devised to explain the complex nature of the BR photo-cycle. Such models include, but are not limited to, the Isomerization-Switch- Transfer (IST) model (Lanyi, 1998b; Haupts et al., 1997; Tittor et al., 1997), the Local-Access model (Lanyi, 1998a; Brown et al., 1998a; Brown et al., 1998b) and the Hydration model (Kandori, 2004).

2.1 The main and branched photocycle of native bacteriorhodopsin with approximate lifetimes at ambient temperature. Photointermediates are shown with the respective absorption maximum in parenthesis (in nanometers).

Regardless of the model, the M, O and Q photostates are demonstrated to be useful targets for numerous biophotonic and bioelectronic applications. Specific discussions of these devices, however, are reserved for section 2.3 of this chapter. The next sections will discuss the main and branched photocycles that result from absorption of a single green photon or sequential green and red photons, respectively.

The main photocycle

The residues within the active site of BR require an exact geometry and set of protonation states for proper functionality (Brown et al., 1994; Tallent et al., 1998; Kusnetzow et al., 1999; Luecke et al., 1999). Figure 2.2 illustrates the quadrupole geometry of the BR active site. This arrangement is well resolved to contain the positively charged Schiff base nitrogen of the chromophore-lysine linkage, two negative aspartic acids and one positive arginine that are all coordinated around three water molecules. Experimental evidence of this arrangement is upheld by a combination of theoretical (Birge and Zhang, 1990; Tallent et al., 1998; Kusnetzow et al., 1999), mutagenic (Duñach et al., 1990; Heberle et al., 1993; Balashov et al., 1995a; Hutson et al., 2000; Shibata et al., 2003) and crystallographic data (see Table 2.1). Perturbation of this system, via chemical or mutagenic manipulation, greatly reduces the ability of the protein to function properly (Kobayashi et al., 1983; Mogi et al., 1988; Heberle et al., 1993; Moltke and Heyn, 1995).

Table 2.1

X-ray crystallographic determinations of the bacteriorhodopsin photointermediates

aProtein Data Bank (http://www.pdb.org)

bThe differences observed between the L-state structures are compared to spectroscopic data and discussed in Lanyi (2004a)

cM1 photostate produced at room temperature

dV49A mutant

eAcid blue form of native bacteriorhodopsin with a resting state at 603 nm

fD85S mutant with a resting state at 603 nm

2.2 The quadrupole geometry of the bacteriorhodopsin active site comprises four residues and three water molecules. The model was created with the Visual Molecular Dynamics software package (http://www.ks.uiuc.edu/Research/vmd/) and using the 1C3W crystal structure (Luecke et al, 1999).

The main photocycle, comprising of the bR, K, L, M, N and O photostates, is a fast event (~ 10–15 ms) that translocates a proton across the membrane (Birge, 1994a). Under continuous illumination, this process is capable of producing and maintaining a proton motive force sufficient for the anaerobic synthesis of adenosine triphosphate (ATP) (Oesterhelt and Stoeckenius, 1973). The high quantum efficiency (ϕ ~ 0.65) of the primary photochemical event makes this process very efficient and a detailed discussion of the photophysics is presented by Stuart and Birge (1996). This absorption event produces an excited state, often recognized as the K intermediate, and involves the isomerization of the all-trans retinal to a 13-cis configuration after the absorption of a photon. Additionally, it should be noted that other photointermediates (i.e. I and J) are reported to precede K with submicro-second lifetimes (Kobayashi et al., 1983; Doig et al., 1991; Shim et al., 2008). Attempts to thermally trap these states, however, have been unsuccessful at temperatures as low as four Kelvin (Birge et al., 1999). Many of the other photostates have been trapped and studied spectrally (Smith et al., 1983; Bressler et al., 1999; Kobayashi et al., 2001; Shim et al., 2008) or via X-ray crystallography (Table 2.1). Hence, these untrappable states can be explained as a vibrationally hot K state that contains a mixture of ground and excited states (Birge et al., 1987).

Proton transport through BR is conventionally divided into five steps (Fig. 2.3). The following discussion highlights the key transfer events that follow photoexcitation of the protein and the reader is advised to investigate the select reviews by Balashov (2000), Lanyi (2000, 2004) and Luecke et al. (2000) for a more detailed discussion of the photocycle. The first proton transfer event occurs during the LM conversion where a proton is transferred from the Schiff base nitrogen to Asp-85 (Metz et al., 1992; Zimányi et al., 1992; Dickopf and Heyn, 1997). Retinal is in a distorted 13-cis, 15-anti configuration during the M state (Schobert et al., 2003). The protein then releases a proton from the proton release complex as a dark reaction. This dark reaction is not represented by an observed photointer- mediate and occurs as the protein undergoes a conformational change from the M→N state (Balashov et al., 1993; Heberle et al., 1993). Debate over the residues involved in the proton release complex, or even its existence, is present in the literature, but mutational studies of the glutamic acids at the extracellular surface demonstrate that Glu-194 and Glu-204 are vital to the structure and function of BR (Richter et al., 1996a; Richter et al., 1996b; Balashov et al., 1997; Dioumaev et al., 1998; Sanz et al., 2001). Water, as a single molecule or complex of molecules, is also essential for proton release (Luecke et al., 1998; Rammelsberg et al., 1998). The third step involves reprotonation of Schiff base nitrogen via Asp-96 (Cao et al., 1991; Cao et al., 1993). Translocation of this proton must span a distance of seven to nine angstroms and it is generally accepted that a hydrogen-bonded network of water molecules is involved (Cao et al., 1991; le Coutre et al., 1995; Sass et al., 2000). The fourth step involves reprotonation of Asp-96 from the cellular cytoplasm and isomerization of retinal from a 13-cis to all-trans configuration (Holz et al., 1989; Otto et al., 1989; Brown et al., 1999). This step coincides with the O state. The final proton transfer event is the reprotonation of the proton release complex via Asp-85 (Richter et al., 1996c). This step reforms the bR resting state, where the protein will remain until another photon is absorbed by the chromophore.

2.3 Proton transport through bacteriorhodopsin. The details of this mechanism are described in section 2.2.1 under ‘The main photocycle’.

The branched photocycle

Access to the branched photocycle is possible by several routes (Popp et al., 1993; Tallent et al., 1998; Birge et al., 1999; Masthay et al., 2002), as illustrated in Fig. 2.4, but the sequential absorbance of green and red photons is the best understood mechanism. When a red photon is absorbed during the O state, the protonated all-trans retinal photoisomerizes to a protonated 9-cis configuration that is unstable in the binding pocket (Popp et al., 1993). The bound 9-cis chromophore is represented by the P state. This state was originally defined as having a λmax ~ 490 nm (Chang et al., 1987), but was later shown to comprise the P1max = 525 nm) and P2max = 445 nm) states that undergo a dynamic equilibrium after photon absorption (Gillespie et al., 2002). Hydrolysis of the Schiff base produces an inactive form of the protein, termed the Q state (λmax ~ 390 nm), that results from the unfavorable 9-cis geometry of the chromophore within the BR binding pocket (Popp et al., 1993). The free retinal is trapped within the protein binding site and, with an activation barrier of ~ 190 kJ mol− 1, the Q state is stable for 7–12 years at ambient temperature (Birge et al., 1999). Reversion of Q to bR is possible by the absorption of a blue photon by the 9-cis chromophore (Dancshazy and Tokaji, 2000).

2.4 Scheme of the various methods for accessing the branched photocycle of bacteriorhodopsin. The Laser Induced Blue Membrane (LIBM) and Deionized Blue membrane (dIBR) are discussed in section 2.4.1 under ‘Experimental pH’. The absorption maximum (in nanometers), chromophore configuration and state of the Schiff base linkage between retinal and Lysine 216 are noted beneath each photostate.

2.2.2 Other retinylidene proteins with the potential for application

Hundreds of type I retinylidene proteins have been reported in recent years, many of which are putative opsins, from all domains of life (Spudich et al., 2000; Adamian et al., 2006; Rusch et al., 2007). We note that type II retinylidene proteins of higher eukaryotes have been investigated for their potential as a biomaterial (see Vsevolodov (1998) and references therein), but their application is limited because many such proteins photobleach upon activation (Shichida, 1986). The mechanism by which these molecules function is also more complex than type I retinylidene proteins, which are often simple ion pumps or channels. As G-protein coupled receptors, many type II retinylidene proteins interact with soluble proteins, by means of their intracellular loop regions, and initiate a cascade mechanism that is yet to be useful in applications, biomimetic or otherwise. The type I retinylidene proteins that demonstrate potential usefulness are described in the following sections.

Eubacterial proteorhodopsins

Proteorhodopsin (PR), a eubacterial photoactive membrane protein, was discovered in 2000 and is identified as a proton pump with a rhodopsin-like topology (Béjà et al., 2000; Rangarajan et al., 2007; Gourdon et al., 2008). Found throughout the oceans of the Earth (Béjà et al., 2000; Béjà et al., 2001; Man et al., 2003; Sabehi et al., 2004; Rusch et al., 2007), all of the identified variants are classified as either blue-absorbing (BPR; λmax ~ 494 nm) or green-absorbing (GPR; λmax ~ 520 nm) based on the relative absorption maximum to one another (Béjà et al., 2001; Wang et al., 2003; Rusch et al., 2007). Variants of both PR sub-families share highly similar primary sequences (~ 80% identity) and exhibit photocycle speeds that differ by an order of magnitude (Béjà et al., 2001; Man et al., 2003); however, only the GPR photointermediates have been well resolved to date (Lakatos et al., 2003; Váró et al., 2003; Huber et al., 2005; Xiao et al., 2005).

Despite literature bias of GPR characterization, the analogous photo- cycles of BR and PR has drawn significant attention for the striking similarities between these proteins (Váró and Lanyi, 1991; Gillespie et al., 2002; Váró et al., 2003). Specifically, the potential applications of PR in protein- based devices are currently boundless because these proteins are both stable and functional when non-natively expressed and detergent solubilized. Bacteriorhodopsin, which exhibits extraordinary photochemical and thermal stabilities, becomes significantly fragile when removed from the native lipids of Halobacterium salinarum (Brouillette et al., 1989; Hendler and Dracheva, 2001; Heyes and El-Sayed, 2002). Thus, the ability of PR to remain functional and highly stable in a non-native lipid environment is a significant advantage over BR.

From a materials standpoint, PR exhibits several strategic advantages over BR. First, GPR exhibits a fast photocycle (~ 15–50 ms), with photoin-termediates that are comparable to those of BR, when suspended in a phospholipid environment (Béjà et al., 2001; Xiao et al., 2005; Bergo et al., 2009). Like BR, however, the photocycle kinetics of GPR are affected by the experimental conditions, as evidenced by the lifetimes shown in Fig. 2.5. No detailed study of the BPR photocycle has been conducted to date. Second, PR exhibits BR-like photophysical properties when suspended in various lipid environments either from the host of non-native expression methods (Béjà et al., 2001; Lörinczi et al., 2009) or detergent solubilization (Kim et al., 2008; Xi et al., 2008). The ability to generate stable detergent-PR suspensions marks a significant advantage over BR, which is structurally and functionally altered without the native host lipids (Dencher et al., 1983; Brouillette et al., 1989; Hendler and Dracheva, 2001; Heyes and El-Sayed, 2002b). We do not suggest that the current photophysical properties of PR are equivalent to those exhibited in the native lipid environment, which is presently unknown, but rather that the detergent solubilized form is comparable to native BR and is currently useful in device applications.

2.5 The photocycle of green proteorhodopsin in a (a) non-ionic detergent (Dioumaev et al., 2002; Váró et al., 2003), or (b) phospholipid environment (Krebs et al., 2002; Xiao et al., 2005; Bergo et al., 2009) at ambient temperature. Lifetimes are shown for select photostates that have been resolved in the cited references. The O* state of the detergent photocycle represents the pR′(O) state, which is structurally identical to the pR resting state, and is only observed by kinetic analysis of UV–vis spectra (Váró et al., 2003) or FT-IR spectroscopy (Dioumaev et al., 2002). Similarly, the M2 state of the detergent photocycle was kinetically resolved from the absorption data at 420 nm (Varo et al., 2003). The dashed trajectories of proton uptake or release represent an approximate assignment of the event.

Many of the proposed PR-based technologies are logical extensions of existing BR-based devices. For example, the holographic (Xi et al., 2008) and photovoltaic (Dioumaev et al., 2003; Tamogami et al., 2009) properties of PR have been reported with similar efficiencies to BR. There are also patents that employ PR in security inks (Jensen et al., 2008), binary optical memories (Stuart, 2008) and solar cells (Delong, 2007). Although the numerous similarities between BR and PR will expedite the development of PR-based devices, much remains to be explored before such devices can become a reality. For example, few studies address the structural nature (i.e. quaternary structure) of PRs. The existing PR structural literature, although sparse, agrees on a rhodopsin-like structure (Béjà et al., 2000; Rangarajan et al., 2007; Gourdon et al., 2008) that can assemble into stacked lamellar sheets of two-dimensional (2D) oligomeric protein arrays when suspended in a cationic detergent (Liang et al., 2007).

Halorhodopsin

Halorhodopsin (HR), discovered in 1980 as a light-driven ion-pump belonging to the protein family of archaeal rhodopsins (Matsuno-Yagi and Mukohata, 1980), has recently emerged as a potentially useful photosensitive protein for device applications. In 2000, Kolbe et al. determined the X-ray structure of HR at 1.8 Å resolution, revealing a 31% sequence identity with BR. Despite the high degree of homology, HR functions as a chloride ion pump in which Cl ions move from the extracellular milieu to the cell cytoplasm. The direction of ion movement, which is opposite of BR, produces an electrochemical ion gradient that enables pH control in H. salinarum (Spudich, 2000; Essen, 2002). Subsequent studies on HRs of H. salinarum and Natronomonas pharaonis determined that HR pumps Cl ions into the cell cytoplasm after illumination with yellow (~ 580 nm) light. The majority of device applications that employ HR (discussed below) use N. pharaonis HR (NpHR) due to its enhanced stability and chloride affinity over Halobacterium species (see Zhang et al. (2007) and references within). However, one of the first applications of HR, the development of an anion-sensitive biosensor by Seki et al. (1994), used halophilic bacteria containing HR.

When NpHR is exogenously expressed in neurons, the photoactive protein can be used to silence neuronal electrical activity by hyperpolarizing the neurons rapidly and reversibly upon illumination with intense yellow light (Lynagh and Lynch, 2010). Arrenberg et al. (2009) generated a transgenic zebrafish expressing enhanced NpHR in order to investigate the silencing of neurons. Concurrently, Tønnesen et al. (2009) studied the use of NpHR in organotypic hippocampal cultures to inhibit excessive hyper- excitability and epileptiform activity.

Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a retinylidene protein that functions as a light-gated non-specific cation channel, which conducts H+, Na+, K+ and Ca+ 2 ions into the cell cytoplasm after exposure to blue light (~ 460 nm). This microbial-type rhodopsin is found in the green algae Chlamydomonas rein-hardtii and is responsible for phototaxis and photophobic responses (Nagel et al., 2003). Although there is low sequence homology to BR, several amino acids that define the retinal binding site and the proton transport network are conserved (Nagel et al., 2005).

In the mid 2000s, ChR2 emerged as one of several light sensitive molecules that could potentially photostimulate excitable cells such as neurons, endocrine, cardiac or skeletal cells (Zhang et al., 2006). Investigation into the engineering aspects of ChR2 revealed that the fast kinetics, high conductance and good temporal resolution of the photoresponse of ChR2 made the photoactive protein a promising candidate for future neuron circuit models (Nikolic et al., 2006). Initial studies by Boyden et al. (2005) demonstrated that illumination of ChR2-expressing neurons with blue light generated action potentials with millisecond precision. Subsequent studies on neurons expressing ChR2 proved that ChR2 could be used rapidly and non-invasively to control neural circuit activity with optical stimulation (see Campagnola et al. (2008) and references within).

The combination of NpHR and ChR2 as a neuroengineering tool was first proposed by Zhang et al. (2007) in order to investigate neural circuit function. The goal was to develop precise neuromodulation technologies for the treatment of neuropsychiatric disorders, which may result from circuit-level effects of malfunctioning neurons. Because the wavelengths of activation for each protein are separated by nearly 100 nm, the neurons, expressing either NpHR or ChR2, could be controlled independently to either drive action potential firing (ChR2-expressing neurons) or suppress neural activity (NpHR-expressing neurons) (Zhang et al., 2007). Recently, Bernstein et al. (2008) genetically expressed ChR2 and NpHR into neuronal cells in the cortex of a non-human primate, a key step for translating previous advances in optogenetic neuromodulation vertebrate and invertebrate therapies to human therapies.

Several applications have emerged for light-based cell stimulation including optogenetic neuromodulation therapy (Zhang and Deisseroth, 2009), a photo-ionic pacemaker (Sergot et al., 2010), an artificial retina (Balya et al., 2008) and restoration of ON/OFF responses in retinas with photoreceptor degeneration (Zhang et al., 2009). The use of both NpHR- and ChR2- expressing excitable cells allows for the facilitation or inhibition of the flow of cations or anions through cellular membranes and results in the hyper-polarization or depolarization of the photosensitized cells. There are several advantages of optical stimulation over electrical stimulation. First, by using light as the stimulus there are no unwanted side effects from extraneous stimulation of adjacent non-targeted neurons. Second, electrodes have limited, sometimes inadequate, mechanical stability as well as issues with migrating away from targeted areas. Third, encapsulation by glial cells over time increases the resistance of the electrode and consequently the voltage needed to reach the targeted cells. For reviews on optogenetic technologies and photochemical tools for controlling neuronal activity see Kramer et al. (2009) and also Baler and Scott (2009).

2.3 Applications of bacteriorhodopsin

An ideal photochromic material should exhibit excellent resistance to chemical, photonic and thermal stress. Bacteriorhodopsin represents such a material because it retains both structural and functional integrity at high temperatures (Jackson and Sturtevant, 1978; Wang and El-Sayed, 1999; Muller et al., 2000; Wang and El-Sayed, 2000) and over a broad range of pH (Brouillette et al., 1987; Kono et al., 1993; Rammelsberg et al., 1998; Balashov, 2000). Furthermore, the protein exhibits increased stability, up to 140°C, when prepared as a dry film (Shen et al., 1993). Such stability is uncommon in most proteins, which denature or become inactive outside of their physiological conditions, and results from several structural features of the protein that include: the semi-crystalline organization of the protein within the membrane bilayer (Henderson et al., 1990; Grigorieff et al., 1996; Heyes and El-Sayed, 2002), cationic cofactors (e.g., Ca2 +, Mg2 +) (El-Sayed et al., 1995; Heyes and El-Sayed, 2001), an αII type helical structure (Krimm and Dwivedi, 1982; Wang and El-Sayed, 1999; Wang and El-Sayed, 2000) and specific interactions with the native H. salinarum lipids (Heyes and El-Sayed, 2002a; Kresheck et al., 1990). (See Heyes and El-Sayed (2003) for a review of how these factors stabilize BR.) These factors have indirectly optimized BR for application as a robust biomaterial by exposing the protein to a brine environment. Nonetheless, the extraordinary stability of BR makes it an excellent biomaterial for which it has found application in a vast array of optical devices (Table 2.2).

Table 2.2

Technologies derived from the bacteriorhodopsin photocycle

Application Type Reference
Artificial retinas Bioelectronic (Miyasaka et al., 1992; Takei et al., 1992; Chen and Birge, 1993a)
Associative memories Biophotonic (Birge, 1990; Hillebrecht et al., 2005)
Dynamic time-average interferometers Biophotonic (Renner and Hampp, 1992)
Holographic correlators Biophotonic (Thoma and Hampp, 1992; Zhang et al., 1995)
Multi-level logic gates Biophotonic (Gu et al., 1996)
Neural-type logic gates Bioelectronic (Mobarry and Lewis, 1986)
Non-linear optical filters Biophotonic (Thoma et al., 1991)
Optical computing Biophotonic (Lewis et al., 1997)
Optical limiters Biophotonic (Huang et al., 2004; Song et al., 1993)
Pattern recognition systems Biophotonic (Hampp et al., 1994)
Photon counters and photovoltaic converters Bioelectronic (Marwan et al., 1988; Sasabe et al., 1989; Hong, 1994)
Photovoltaics Bioelectronic (Hong, 1997b)
Picosecond photodetectors Bioelectronic (Rayfield, 1989; Rayfield, 1994)
Random access thin film memories Biophotonic (Lawrence and Birge, 1984; Schick et al., 1988; Birge et al., 1989)
Reversible holographic media Biophotonic (Vsevolodov et al., 1989; Hampp et al., 1990)
Spatial light modulators Biophotonic (Birge et al., 1990; Song et al., 1993)
Two-photon volumetric memories Biophotonic (Birge et al., 1994; Vought and Birge, 1999; Stuart et al., 1996; Hillebrecht et al., 2005; Birge et al., 1990; Birge, 1992)

The proposed BR-based devices are defined as either biophotonic or bioelectronic. Biophotonic devices utilize the photophysical properties of the transient intermediates produced during the BR photocycle (see section 2.2.1). Recall that the protein translocates a proton after activation and, under prolonged illumination, is capable of creating a proton motive force that sustains anaerobic respiration (Oesterhelt and Stoeckenius, 1973). This force results in a physical charge displacement that translates into an electrical signal that is useful in bioelectronic devices (Hong, 1997). Select biophotonic and bioelectronic devices are discussed in the subsequent sections and the reader is encouraged to explore the cited references for specific details pertaining to each application.

2.3.1 Associative memories

The encoding, manipulation and retrieval of data from biomacromolecules defines the field of biomolecular electronics. Biological molecules and proteins offer inherent advantages over conventional mechanical and computer engineering. These advantages stem from natural selection. Evolutionary processes select for organisms that are efficient and robust machines, able to carry out specific functions in nature. Bacteriorhodopsin, a protein with a high photochemical efficiency, thermal stability and cyclicity, serves as an excellent example of a protein that is useful for application in biomolecular devices (Stuart et al., 2003). Unfortunately, nature has little stake in optimizing proteins for non-native functions and environments. Chemical modification or mutagenesis is necessary for these biomolecules to function in applied technologies such as holographic associative memories (Wise et al., 2002). The most successful of these applications is the Fringemaker holographic interferogram developed by Juchem and Hampp (Juchem and Hampp, 2000; Hampp and Juchem, 2000).

Associative memories function differently from serial memories that dominate current computer designs. These memories take an input ‘image’, and independently ‘scan’ the entire memory for a page of data that matches the input. In most implementations, the memory finds the closest match when a perfect match is not available. Finally, the memory will return the memory page that fulfills the matching criteria. The most sophisticated associative memories allow for variable-sized blocks, which make the association process adaptable to the amount of information returned. Because the human brain operates in a neural, associative mode, the implementation of large capacity associative memories will likely be a necessary component of any computer architecture that achieves artificial intelligence (Birge, 1990; Birge et al., 1999).

Optical associative memories implement memory recall by using Fourier holographic association (Birge et al., 1999). An example of the optical configuration of a protein-based optical associative memory is presented in Birge et al. (1999). The use of PRs (Xi et al., 2008) and BR (Hampp et al., 1994) in holographic applications is best explored in terms of the refractive index changes that can be generated by converting the protein from the resting state to a blue-shifted intermediate (e.g. the M or Q photostate) (Fig. 2.6). One of the quintessential devices for holography, developed by Nikolai Vsevolodov, the principle scientist of Starzent, uses BR in a prototype holographic memory (Vsevolodov, 1998).

2.6 The relative holographic efficiency (thin line) is plotted for 50% conversion of the bR resting state (thick line) to either the (a) Q state, or (b) M state. Dashed lines represent both the M and Q states in the respective figures. The bR→Q state conversion yields a maximum holographic efficiency of 8.0% at 665 nm, whereas the bR→M state conversion yields a maximum holographic efficiency of 6.3% at 670 nm. In general, real-time holographic associative processors or pattern recognition systems use the M state while long-term holographic associative memories use the Q state. All spectra represent data collected at ambient temperature.

Historically, the M state has gained the most attention for associative memories, and some of the earliest applications of BR in devices utilized the ability of the protein photochemically to convert from the resting state to the M state (Birge et al., 1992). This transition results in a stable, but short-lived binary system that allows for real-time holography. Additionally, the M state has a λmax of 410 nm, which is important because it induces a large change in the refractive index of the medium following photoconversion. The bR/M holographic efficiency is calculated using the Kramer’s Kronig transformation (Birge et al., 1991; Gross et al., 1992). Because the M state is not permanent, holograms made with the bR/M transition will only last as long as the M state lifetime and the application of this state is limited to real-time dynamic holography.

The Q state has also been considered for holographic memories because, when formed, it is stable for years, and it has a blue-shifted λmax (~ 380 nm) in comparison to the M state, as illustrated in Fig. 2.6 (Birge et al., 1999). Furthermore, devices based on the Q state remove the time-sensitive nature of devices based on the M state because the M state is only stable for minutes (Stuart et al., 2007). Formation of the Q state occurs via a sequential two-photon event that is described in section 2.2.1 under ‘The branched photocycle’ and methods for enhancing Q state formation are discussed below in section 2.4.3.

Manipulation of the M and Q photostates allows for the generation of more efficient memory systems. Alterations in pH, temperature, solvent environment, chromophore configuration and the addition of chemicals are some of the ways in which the protein is modified (Schmidt et al., 1998; Stuart et al., 2002). Mutagenesis of BR to enhance M state formation usually targets residues involved in the proton transfer from the intracellular surface to the Schiff base (Wise and Birge, 2004). The M → N transition is associated with reprotonation of the Schiff base, and therefore disruptions, such has a decrease in the pH of the system, will stabilize the M state. Site-directed mutagenesis by Hampp et al. (Hampp et al., 1990; Hampp, 2000b) and Holz et al. (1989) reveals that the mutant D96N yields a twofold increase in the diffraction efficiency of the protein as well as an increase in the photosensitivity of thin films prepared with the mutant in comparison to wild type. Additionally, the lifetime of the M state increases from approximately nine milliseconds in wild type BR to nearly 750 ms in the D96N mutant (Pandey et al., 1999).

2.3.2 Volumetric memories

Three-dimensional (3D) memories store information in a volumetric memory medium and offer as much as a 1000-fold improvement in data storage capacity for a given enclosure size. However, optical and reliability considerations of these devices tend to reduce the comparative advantage factor to values closer to a 300-fold improvement. The branched-photocycle architecture discussed herein is made possible by an unusual photochemical characteristic of BR that provides the ability quickly and reliably to store and retrieve information. A prototype device for this memory is shown holding a ‘data cuvette’ in Fig. 2.7.

2.7 Two versions of the sequential two-photon volumetric memory (a) in the form of the original prototype, and (b) as built for the United States Air Force. The protein is immobilized in a polyacrylamide matrix within a sealed three-milliliter cuvette. Details of the theory and methods for preparing these memories are discussed by Birge et al. (1999) and Stuart et al. (2002).

Exploitation of the branching reaction, which was introduced in section 2.2.1 under ‘The branched photocycle’, is achieved by assigning the resting state (bR) to bit 0 and both P and Q photostates to bit 1 (Fig. 2.8). This model indicates the wavelength maximum (in nanometers), the configuration and the protonation state of the chromophore directly underneath the intermediate label. The 3D aspect of this branching reaction derives from the fact that the green (λmax ~ 570 nm) paging beam is orthogonal to the red (λmax > 600 nm) write beam (Stuart et al., 1996; Stuart et al., 2000; Stuart et al., 2003). To be specific, the paging beam selects a thin page inside the memory medium, and the write beam arrives a few milliseconds later with the data imposed upon it via a spatial light modulator. The red beam interacts with the O state and converts the irradiated voxels, and only the irradiated voxels, from O→P. The P state then spontaneously converts to the Q state, and it is Q that stores the data in a format that is long-term and shifted in wavelength space so that they are not erased during other read-write cycles. Those voxels within the page that were not irradiated with the write beam continue the photocycle and return to the bR resting state. Thus, a page of data can be written without disturbing data written elsewhere in the volumetric memory medium. A write process proceeds in a comparable fashion, but the spatial light modulator turns on all of the pixels but at a very low level sufficient to image the page onto a detector. The process is described in detail in Birge et al. (1999).

2.8 Branching reaction of bacteriorhodopsin. This model indicates the wavelength maximum (in nanometers), the conformation and the protonation state of the chromophore directly underneath the intermediate label.

The key requirement of a protein to be used in a sequential two-photon Q-based memory is efficient O→P photochemistry. The native protein is quite inefficient at this process, and memories based on the native protein require 100–200 mW lasers to carry out the conversion process. By using directed evolution, the efficiency of the Q state photochemistry has been extensively enhanced to where it is useful in volumetric memories. Optimization of the Q state by this method is described in section 2.4.3.

2.3.3 Photovoltaics

The native function of BR is the light-activated construction and maintenance of a proton gradient across the membrane that drives ATP-synthase under anaerobic conditions (Oesterhelt and Stoeckenius, 1973; Oesterhelt, 1998). This translocation event results in a surplus of positive charge, up to 4 pH units on the extracellular membrane surface (Calimet and Ulmann, 2004), that can be measured as an electrical response to a light stimulus (Hong, 1994). Magnification of the electrical signal occurs when the protein is condensed into a film on an electrically conductive surface (Váró, 1981; Váró and Keszthelyi, 1983). Proper orientation of the protein within a film, however, enhances both the signal and reproducibility of the experiment. Methods for orienting the protein films include, but are not limited to: sol-gel encapsulation (Chen et al., 1991), antibody-mediated orientation (Koyama et al., 1994), electric field sedimentation (Váró and Keszthelyi, 1983), Langmuir-Blodgett deposition (Miyasaka and Koyama, 1992) and electrostatic layer-by-layer adsorption (He et al., 1998). The duration and magnitude of the photovoltaic signal are sensitive experimental conditions (e.g. light intensity, humidity). A comprehensive review by Hong outlines the conditions, via electrical circuit models, that control the photoelectric signal of BR (Hong, 1999).

Illumination of BR films results in one of two photovoltaic responses: a fast photoelectric pulse or a slow differential response. The fast event is observed at high temporal resolution following flash photolysis (Fig. 2.9). This signal is conventionally divided into the B1, B2 and B3 components.

2.9 Fast electrical response that is produced by flash photolysis of a bacteriorhodopsin film that is sedimented onto glass coated with indium-tin oxide.

The B1 signal is a sharp negative voltage spike with a rise time of ~ 6 ps and is aligned with the photoisomerization of the retinal chromophore from 13-s-trans to 13-s-cis (Xu et al., 2003). The relative magnitude of the negative signal is controlled by the molecular motion of Arg-82 and can theoretically be mediated via substitution with other amino acid residues (Xu et al., 2003). Conversion of this negative signal to the positive B2 phase is associated with the K→L→M photointermediates of the BR photocycle (Fig. 2.1) (Keszthelyi and Ormos, 1980; Xu et al., 2003). Enhancing the B2 signal is accomplished by manipulation of the M state lifetime. The B3 signal, which is responsible for the long tail in the signal, is associated with the M→N→O→bR portion of the photocycle (Keszthelyi and Ormos, 1980).

The slow differential responsivity of BR thin films results from long periods of illumination (Fig. Fig 2.10). Comprising transient positive and negative signals, each of which correlate with the onset and termination of the light stimulus, this amalgamated photoresponse presents as either a square wave or a differential response (Hong, 1997). Realize that the shape represents the creation and maintenance of a displaced charge that is directly reflected in the signal. Symmetrical positive and negative transitions are produced when the protein is uniformly aligned and is capable of maintaining, or storing, the charge at one surface of the film. This phenomenon has been occasionally termed chemical capacitance and has been likened to an electrical circuit (Hong, 1997).

2.10 Differential photovoltage produced by a bacteriorhodopsin film that was exposed to 150 ms of continuous illumination of filtered (550–600 nm) light.

2.3.4 Photosensors and artificial retinas

The photoelectric properties of BR, which are derived from the ability of the protein to generate a pH gradient upon light absorption, make the protein a viable candidate for photoelectric sensors (Birge, 1990; Haronian and Lewis, 1991; Takei et al., 1992; Boyer et al., 1995; Knopf et al., 2009), motion detectors (Miyasaka and Koyama, 1993; Fukuzawa, 1994; Yao et al., 1997) and complex artificial retina devices (Miyasaka and Koyama, 1992; Chen and Birge, 1993; Hong, 1997). The proton pumping capability of BR transports a proton roughly five nanometers from the cytoplasmic to the extracellular milieu to generate a pH gradient across the membrane (Oesterhelt and Hess, 1973; Birge, 1990). This photoelectrical signal can be amplified when the protein is condensed into a thin film (Váró, 1981; Váró and Keszthelyi, 1983). The resulting photovoltage is complex in nature and can be supported by an embedded electrolyte-containing layer within a photoelectric cell (see detailed discussion in section 2.3.3) (Hampp, 2000a). The intrinsic properties of BR that make it uniquely qualified for use in photoelectric devices include: (1) long-term thermal and photochemical stability, (2) high forward and reverse quantum yields, which allow for activation at low light levels, (3) picosecond photochemical response times, (4) wavelength-independent quantum yields, (5) generation of photoelectric signal that has an opposite polarity for the forward and reverse photo-reactions, (6) differential responsivity that mimics in vivo photoreceptors under certain conditions and (7) the ability to reproducibly form oriented thin films or volumetric cubes of the protein (Birge, 1990; Chen and Birge, 1993; Tukiainen et al., 2007).

Photosensors

One of the earliest applications of the photoelectric properties of BR was implemented by Trissl et al. in the development of a super-fast photodetection device (Trissl, 1987; Trissl et al., 1989). Shortly after, Haronian and Lewis (1991) demonstrated that the electrical effects of BR are analogous to conventional neural network architectures through the development of a reprogrammable BR-based neural network. In conventional systems, the energy (i.e. voltage) is supplied by the neuron, whereas in the device proposed by Haronian and Lewis (1991) the BR molecule, upon photoexcitation, supplies the energy. This neural network device was one of the first examples of electrooptical erasable memory and also encouraged the development of BR-based image sensing (Haronian and Lewis, 1991).

While investigating the strange behavior of BR at the electrode-electrolyte interface, Boyer et al. (1995) discovered the possibility of color discrimination by BR-based photosensors. Similar to the differential photosensitivity of BR (see section 2.3.3), forward and reverse photocurrents, having opposite signs, were measured on the green absorbing ground state (bR570) and a structurally and spectrally modified bR480. The altered pigment (bR480) was generated by the addition of halogenated anesthetics and maintained full functionality. Results of these experiments indicated that BR readily self-orients itself at the electrode interface (Boyer et al., 1995).

Motion detection and edge detection

Neural networks are fundamentally characterized by their ability to elicit excitatory and inhibitory responses. In retinal ganglion receptive fields, the ability to detect a sharp intensity variation in image space, i.e. edge detection, as well as the ability to detect motion of objects is feasible due to the different response times for the excitatory and inhibitory regions of the receptive fields. Significant efforts have been made to develop a biomaterial that can mimic the inherent ability of the retinal neural network. In the early 1990s, several reports of optoelectric devices using BR demonstrated that receptive fields of BR are capable of excitatory and inhibitory responses as well as edge detection at zero crossings (Takei et al., 1992; Martin et al., 1997; Yao et al., 1997; Yang and Wang, 1998). Additionally, a wet-type 256-pixel artificial photoreceptor created from immobilized thin films of BR at the solid–liquid interface of an electrode surface was used to demonstrate the ability of BR to detect motion and edge information of images in real time (Miyasaka and Koyama, 1993). This unique ability of BR to detect motion is a result of a differential responsivity to light intensity (see section 2.3.3) (Hong, 1997). Although the aforementioned BR-based motion sensors were successfully shown to sense the motion and direction of movement from an object, the sensors were unable to detect the displacement of a moving object without the use of an external memory device. The image-recording capability of BR was first demonstrated by treating BR thin films with a high pH buffer. A key advantage of the high pH BR sensor was the ability of the sensor to detect an object’s current and previous position without the use of an external memory device. The authors noted this unique advantage of a BR-based motion sensitive position sensor could find useful application in robotic vision (Fukuzawa, 1994).

Artificial retinas

Protein-based artificial retinas have been proposed based on the use of BR, which converts light into an electrical signal, to activate the retinal ganglion or bipolar cells. The complex photovoltaic signal produced by BR during its photocycle has been reported to be as large as 5 VDC (Crittenden et al., 2003; Zhang et al., 2003), while experimental data show that a signal of only 1.6 VDC can stimulate nerve impulse (unpublished data). Through genetic engineering it is believed that the photovoltaic signal of BR can be enhanced three-to-five fold. The proposed motion-sensitive artificial retinas of Miyasaka (Miyasaka and Koyama, 1992) and Birge (Chen and Birge, 1993) use the differential light responsivity of BR to sense an object’s motion as well as the direction of movement.

Color vision is a result of the combination of three types of cone pigments in humans and animals. Once the visual pigments absorb photons, color may be assigned by comparing the signals from the three cone pigments. Specific details about color vision and BR-based color sensitive biosensors are discussed by Hong (1997, 1999), Lensu (Lensu et al., 2007; Tukiainen et al., 2007), Ogawa (1990) and Martin et al. (1997). Recently, a flexible BR-based photodetector and imaging array, which provides a substantial advantage over previously developed photosensors, was developed by Knopf et al. (2009). Additionally, intelligent artificial retinas with color sensitivity have been proposed by using a combination of native BR with BR analogs that contain modified chromophores (see section 2.4.2) (Frydrych et al., 2000). For a detailed review on color vision and BR-based color sensitive biosensors see Ogawa (1990), Lensu et al. (2007) and references within.

2.3.5 Chemical sensors

Research groups throughout the world are working to develop biosensors with an increased sensitivity and affinity for the detection of biomolecules, including olfactory proteins, DNA and toxic agents involved in biological weapons (Delahanty and Ligler, 2002; Fivash et al., 1998; O’Shannessy et al., 1992). Bacteriorhodopsin is currently being examined as a biomaterial for the aforementioned application (Bryl and Yoshihara, 2001). The protein is shown to have sensitivity to a variety of hydrophobic, polar and charged compounds (Nakagawa et al., 1994; Taneva et al., 1995; Boucher et al., 1996; Lanyi and Luecke, 2001; Heyes et al., 2002). Through molecular modeling of BR with a variety of ligands, specific residues can be targeted for muta- genesis. Mutants can then be created and experimentally tested to generate a library of biomolecules that are capable of chemical detection.

Time-resolved UV-visible spectroscopy is used to monitor BR in response to various concentrations of the target ligand (Fig. 2.11b). The M and O states are used to screen BR for chemical responsivity because these photochemical states are most affected by various chemical additives (Lin et al., 1992; Nakagawa et al., 1994; Lanyi and Luecke, 2001; Heyes et al., 2002). Mutants that show the significant sensitivity are then selected for application in biological sensor devices.

2.11 The three stages involved in the preparation of bacteriorhodopsin for device applications. Phase I involves the genetic engineering of the bacterioopsin (bop) gene. The bop gene is engineered into the pBA1 plasmid, which contains both ampicillin and mevinolin resistance. Transformation of the plasmid into Escherichia coli, a shuttle vector, generates multiple copies of the construct. Escherichia coli is chosen for this step because it reproduces exponentially faster (18 minutes at 37°C) than the native organism, Halobacterium salinarum (18 hours at 40°C). Incorporation of the bop gene into H. salinarum is critical for proper expression of bacterioopsin and is accomplished by transforming the resulting purified DNA into the MPK409 strain of H. salinarum. Next, cells are plated on 5-FOA, which selects for recombinants because the MPK 409 cell line lacks the ura3 cassette. Non-recombinants will die on 5-FOA because they will metabolize the 5-FOA into a toxic byproduct. The details of the mutagenesis process are described in Ni et al. (1990), Wise et al. (2002) and Hillebrecht et al. (2004). Phase II involves the expression of the protein in H. salinarum, which is vital to the structure and function of bacteriorhodopsin (Heyes and El-Sayed, 2002). Recipes for the media and conditions for optimal expression and purification of bacteriorhodopsin are described by Hillebrecht et al. (2004). Phase III involves the biochemical and biophysical characterization of bacteriorhodopsin. Examples of such characterization are: (a) light/dark adaptation (Becher and Cassim, 1976; Dencher et al., 1983; Duñach et al., 1990); (b) temporal measurements of the transient photostates that result from photoactivation (Zimányi et al., 1989; Váró and Lanyi, 1991); and (c) analysis of the retinal configurations within the protein sample (Scherrer et al., 1989; Gillespie et al., 2005).

2.4 Enhancing bacteriorhodopsin for device applications

Bacteriorhodopsin is prepared as large membrane fragments, of gigadalton size, and is commonly referred to as the purple membrane. Comprising only BR and lipids with a 10 : 1 lipid-to-protein ratio (Dracheva et al., 1996; Renner et al., 2005), this structure is approximately 0.5–1.0 μm in diameter and five nanometers thick (Henderson, 1975). This feature makes the protein easy to purify in high yield (~ 20–30 mg L− 1) without the use of expensive chemicals or instrumentation (Oesterhelt and Stoeckenius, 1974; Becher and Cassim, 1975; Lorber and DeLucas, 1990). A general scheme of the protein preparation method is shown in Fig. 2.11. Modulating the photo- physical properties of BR is possible by several methods. The most common methods for modulating these properties are through chemical manipulation of the bulk structure, substitution of the chromophore with synthetic chromophore analogs and genetic engineering of the native protein.

2.4.1 Chemical manipulation

Chemical modification of BR is accomplished by any number of methods that expose the protein to a non-native solute or solvent, solubilize the protein in a detergent, reconstitute BR in an alternate lipid environment or modify the organic chromophore. These conditions will be discussed in the subsequent section with exception to the studies that integrate synthetic retinal analogs within the active site of BR. This last condition represents a large subfield of BR optimization and will be reviewed in section 2.4.2.

Lipid environment

Bacteriorhodopsin is natively expressed by Halobacterium salinarum, a halophilic archaea, when oxygen levels become too low to sustain respiration (Oesterhelt and Stoeckenius, 1973). The methods for cultivating the protein in this way recreate such an environment in the laboratory (Oes-terhelt and Stoeckenius, 1974; Becher and Cassim, 1975; Lorber and DeLucas, 1990). Although H. salinarum is slow growing, with a doubling time of 18 hours at 40°C, expression of the protein within the native membrane is absolutely necessary for proper production of this biomaterial. Non-native expression, via Escherichia coli (Braiman et al., 1987; Dunn et al., 1987; Hackett et al., 1987; Karnik et al., 1987; Mogi et al., 1987; Nassal et al., 1987), and reconstitution of BR in non-native lipid structures e.g. liposomes (Sugihara et al., 1982; Torres et al., 1995; Kalaidzidis et al., 1999), unilamellar vesicles (Piknova et al., 1993), mixed micelles (Allen et al., 2001; Brouillette et al., 1989; Booth et al., 1997), and detergent suspensions (Dencher and Heyn, 1978; London and Khorana, 1982; Muccio and DeLucas, 1985; del Rio et al., 1991; Massotte and Aghion, 1991) has been attempted. However, the function and structure of BR are significantly altered without the native archaeal lipids (Dencher et al., 1983; Brouillette et al., 1989; Milder et al., 1991; Dracheva et al., 1996; Hendler and Dracheva, 2001; Heyes and El-Sayed, 2002b).

Nanodiscs are a recently developed technology that allows the isolation of membrane proteins within a soluble discoidal structure (see Nath et al. (2007) and references therein). These self-assembling structures offer a novel tool for stabilizing membrane proteins in a phospholipid environment and are quickly finding application with many membrane proteins that include bacteriorhodopsin (Bayburt et al., 2006), cytochrome P450 (Denisov et al., 2007), G-protein coupled receptors (Bayburt et al., 2007) and others (Alami et al., 2007; Bayburt and Sligar, 2009; Borch and Hamann, 2009). Furthermore, isolation of membrane proteins within a nanodisc has minimal effects on the physical properties (e.g. fluidity) of lipids (Shaw et al., 2004; Denisov et al., 2005).

The implementation of these structures in biotechnology is an obvious and simple transition because these structures readily adsorb to surfaces such as glass or mica (Carlson et al., 2000; Goluch et al., 2008; Vinchurkar et al., 2008) and are easily modified with affinity tags (Marin et al., 2007; Shaw et al., 2007). Utilization of such features also offers a means of controlling the protein structure in a realistic and minimally invasive manner. Specifically, BR has been removed from the native purple membrane, an oligomeric structure of gigadalton size, and incorporated into nanodiscs as a monomer and a trimer (Bayburt and Sligar, 2003; Bayburt et al., 2006). Recall that disruption of the native purple membrane lipids, by detergent solubilization or non-native protein expression, often results in undesirable alteration of the photophysical properties of BR (Dencher and Heyn, 1978; Dencher et al., 1983; Massotte and Aghion, 1991). Functional BR was confirmed to exist within nanodisc assemblies, however, by the presence of active trimers as exemplified by the characteristic exciton splitting in visible circular dichroism spectra (Cassim, 1992).

Experimental pH

Altering the environmental pH, which is the simplest form of chemical manipulation, results in three predominant forms of the photocycle (Fig. 2.12). The main photocycle exists between pH 6 and 9.5 and proton transport under these conditions is discussed in section 2.2.1. Application of the photophyscial properties around neutral pH is discussed in section 2.3. Acidification of the protein produces a blue bacteriorhodopsin (bBR) membrane that does not translocate any protons and alters the photophysical properties of BR (Mowery et al., 1979; Maeda et al., 1981; Liu and Ebrey, 1987; Váró and Lanyi, 1989; Moltke and Heyn, 1995). This pigment results from a red shift of the absorption maximum, from 568 nm to 603 nm, that is controlled by protonation of Aspartic acid 85 (Balashov et al., 1995a; Balashov et al., 1996). An acid purple form of the protein is formed when the pH is below 2 and Cl ions are present (Váró and Lanyi, 1989; Logunov et al., 1996; Tokaji et al., 1997). Sulfuric acid is preferentially used over hydrochloric acid for accurate investigations of the acidified bBR membranes (Moltke and Heyn, 1995; Okumura et al., 2005). When illuminated under a specific set of conditions, the acid bBR membranes will produce a 9-cis photoproduct (Maeda et al., 1980; Fischer et al., 1981; Maeda et al., 1981). One should note, however, that this form of BR is less thermally stable and precipitates (Kresheck et al., 1990). The acidified protein is often encased in a matrix (e.g. gelatin, agarose) to prevent precipitation. Deion- ization or mutagenesis of native BR will produce soluble protein that offers analogous photophysical properties to the acid bBR membranes (Hampp et al., 1992; Tallent et al., 1996; Tallent et al., 1998; Millerd et al., 1999). Deionized bBR membranes will be discussed in the following section.

2.12 Modulation of the bacteriorhodopsin photocycle via alteration of the experimental pH. The protonation state of Aspartic acid 85 (Asp-85), Aspartic acid 96 (Asp-96), the Schiff base nitrogen of the chromophore-protein linkage and proton release complex (PRC/X) are defined for each experimental condition. The pKA of each residue or complex is described in the resting (bR) state. Below pH 6, which is close to the pKA of the PRC during the M state (Lanyi and Varo, 1995), proton release is delayed until the O→bR transition (Zimányi et al., 1992). A summary of the photophysical properties of bacteriorhodopsin is listed at the center of each photocycle. The values for BR at alkaline pH are nearly identical to those at neutral pH. Image was adapted from (Balashov, 2000).

For comparison, an alkaline form of the photocycle occurs above pH 9.5 where no fast proton release is observed (Kono et al., 1993). Application of this photocycle in devices has yet to be realized because of the truncated photocycle. Upon illumination, the fast formation of the M state is observed (~ 1 μs) with minimal, if any, formation of an O state. This truncation of the photocycle is reported to result from a slow reprotonation of Asp-96 and deprotonation of the proton release complex (often referred to as PRC or X′) (Váró and Lanyi, 1990; Chizov et al., 1992; Govindjee et al., 1996; Richter et al., 1996b). The residues involved in the proton release complex are yet to be absolutely resolved, but significant evidence indicates that several amino acids are involved in proton release during the BR photocycle (Balashov et al., 1995b; Balashov et al., 1996; Bressler et al., 1999; Li et al., 2000).

Deionized membranes

The biological cations that associate with the purple membrane, the oligo-meric structure of BR, are vital for the structural (Cladera et al., 1988; Kresheck et al., 1990) and functional (Chronister and El-Sayed, 1987; Tallent et al., 1998) properties of BR. The surface charge effects and functional changes of cation binding, both monovalent and divalent, to BR have been well characterized experimentally (Chang et al., 1986; Duñach et al., 1988; Alexiev et al., 1994; Birge et al., 1996; Váró et al., 1999; Sanz et al., 2001) and theoretically (Szundi and Stoeckenius, 1987; Szundi and Stoeckenius, 1989). Native BR binds approximately 4 mol Ca2 + and Mg2 + per mol BR at pH 6 (Chang et al., 1985; Chang et al., 1986; Chang et al., 1987) and two high affinity sites (KD = 0.25 and 35 μM) have been identified from isothermal titration calorimetry experiments with Ca2+ (Váró et al., 1999). Displacement of these cations produces a deionized form of the protein (dIBR) that exhibits many similar photophysical properties to the acid bBR membranes (Smith and Mathies, 1985; Chronister and El-Sayed, 1987; Váró and Lanyi, 1989). The properties of the acidified protein are discussed in the previous section ‘Experimental pH’. Deionization of BR is accomplished via acidification (Váró and Lanyi, 1989), laser induction (Masthay et al., 2002) or treatment with ion-exchange resin (Váró et al., 1999), sugar (Rhinow and Hammp, 2008) or chelating agent (Chang et al., 1987; Tallent et al., 1998). An extensive review of the effects of pH and cation binding to the surface charge of the purple membrane is found in Jonas et al. (1990).

Photoexcitation of dIBR with red light (> 640 nm) produces a pink membrane (λmax ~ 490 nm). This photoproduct contains 80% 9-cis retinal (Chang et al., 1987) that is produced in a yield comparable to the 9-cis of the acid blue membranes (Maeda et al., 1980; Fischer et al., 1981) and is similar to the P state of the branched BR photocycle (see ‘The branched photocycle’ in section 2.2.1). Formation of the 9-cis photoproduct is ideal for application in optical memories (see sections 2.3.1 and 2.3.2). Fabrication of dried thin films, comprising dIBR and polyvinyl alcohol, enhances the photochromic properties of the biomaterial (Tallent et al., 1996). Encapsulation of dIBR in a polymer matrix also precludes aggregation of the protein if the pH becomes acidic. No formation of a hydrolyzed 9-cis chromophore is reported in these films, however, and is presumably due to the dehydration of the material. Hydrolysis of the 9-cis chromophore is, by nature, dependent upon hydration of the active site (Popp et al., 1993).

2.4.2 Synthetic retinal analogs

Significant efforts have been made chemically to modify the photoactive chromophore, all-trans retinal, to optimize the properties of BR for device applications (Singh and Hota, 2007). By altering the chromophore structure, the spectral and photochemical characteristics (e.g. absorption maximum, photocycle speed, proton transportation) of native and mutant BRs can be fine-tuned for photochromic, electrochromic and holographic technologies. A summary of the process for developing and fine-tuning BR analogs with modified chromophores is shown in Fig. 2.13 (Khodonov et al., 2000). To date there are over 30 different retinal chromophores available in the literature with modifications to the retinal moiety including: removal of the β-ionone ring, demethylation, lengthening of the polyene chain at the β-ionone ring and locking the retinal into specific configurations (Fig. 2.14) (Vsevolodov, 1998; Singh and Hota, 2007). While most modifications to the retinal have provided valuable insight into the protein/chromophore interaction (Balogh-Nair et al., 1980; Nakanishi et al., 1980; Kakitani et al., 1983; Muradin-Szweykowska et al., 1984; Lugtenburg et al., 1986; Spudich et al., 1986; van der Steen et al., 1986; Zingoni et al., 1986; Drachev et al., 1989; Asato et al., 1990; Liu et al., 1991; Liu et al., 1993; Nakanishi et al., 1995; Yan et al., 1995; Druzhko et al., 1998; Weetall et al., 2000; Haacke et al., 2002; Singh and Manjula, 2003; Bismuth et al., 2007) or the structure/function of the light-absorbing chromophore within the binding site of BR (Marcus et al., 1977; Cookingham et al., 1978; Marcus and Lewis, 1978; Bayley et al., 1981; Huang et al., 1982; Crouch et al., 1984; Crouch et al., 1985; Schiffmiller et al., 1985; Boehm et al., 1990; Büldt et al., 1991; Bhattacharya et al., 1992; Delaney et al., 1995; Druzhko et al., 1996; Tuzi et al., 1996; Rousso et al., 1997; Weetall et al., 1997; Sakamoto et al., 1998; Weetall et al., 2000; Aharoni et al., 2003; Laptev et al., 2008; Gross et al., 2009; Khitrina et al., 2009; Das et al., 1999), few synthetic analogs have been used in device applications. The most promising BRs containing retinal analogs for optoelectronic devices are discussed below.

2.13 Procedure for the preparation of bacteriorhodopsin analogs (adapted from Khodonov et al. (2000)).

2.14 Structures of retinal and synthetic analogs. (1) all-trans retinal, (2) 4-keto retinal, (3) A2-retinal, (4) 13-desmethyl retinal, (5) locked 6-s-trans retinal, (6) locked 6-s-cis retinal, (7) 12,14-ethanoretinal, (8) 3,7,11-trimethyl-2,4,6,8-dodecatetraenal, (9) phenylretinal, (10) 4-methoxyretinal, (11) all-trans 14-fluororetinal, (12) (2Z,4E)-3-Chloro-5-(1H-indol-3-yl)-2,4-pentadienal.

Bacteriorhodopsin containing a 4-keto retinal or the more commonly labeled 4-keto BR has found great application in the area of photochromic and electrochromic materials (Druzhko et al., 1995; Druzhko and Chamorovsky, 1995; Khodonov et al., 2000). The 4-keto retinal analog contains an elongated polyene chain extending from the S-ionone ring (see analog 2 of Fig. 2.14). The resting state of 4-keto BR displays a single absorption maximum (~ 500 nm) that progresses into three blue-shifted absorption maxima, at 390, 420 and 440 nm, after photoexcitation. These peaks have been described as three distinct M-like states and are suggested to be intermediates in the three photocycles of 4-keto BR (Barmenkov et al., 2000). In addition to containing multiple photocycles, 4-keto BR displays photocycle kinetics roughly three times slower than that of native BR. These properties of 4-keto BR may allow for longer storage time of optical information and gives the BR analog a distinct advantage over its native protein counterpart (Druzhko et al., 1995).

The combination of 4-keto BR (λmax = 505 nm), native BR (λmax = 568 nm) and A2 BR (BR containing a 3,4-didehydro retinal, λmax = 590 nm) has shown application in color detection (Lensu et al., 2004; Frydrych et al., 2005) and in a color-sensitive retina (Frydrych et al., 2000). The A2-retinal contains an elongated polyene chain within the β-ionone ring and the A2-BR analog retains optoelectronic activity, similar to 4-keto BR. By altering the absorption maximum of the photocycle states, color sensitivity can be achieved. The three-protein component system is analogous to the color system within the retina as well as the RGB color display system commonly employed in electronic displays (Lensu et al., 2004).

Red-shifted BR pigments have become of particular interest for device applications because they would allow for the use of inexpensive semiconductor lasers as part of the optoelectronic device. Several chromophore analogs (e.g. analog 3 of Fig. 2.14) have been prepared that red-shift the absorption maximum while maintaining the photochromic activity of the BR analog (Muthyala et al., 2001; Druzhko et al., 1998). Additionally, BR analogs containing aromatic rings and heterocyclic rings (e.g. analogs 5, 6, 7 and 12 of Fig. 2.14) have been introduced for potential enhancement of BR-based electronic applications (see Singh et al. (1996), Singh and Manjula (2003), Hota and Singh (2007) and references within).

2.4.3 Genetic engineering

Protein engineering is a necessary step for the successful implementation of BR in applied technologies. Although nature has provided a robust photoactive protein, optimization of native BR is required for application as the primary photochromic material in non-biological environments. In most cases, the focus of mutagenesis is the optimization of the photo- intermediates, M and O, and the branched photoproduct Q, a characteristic rarely found in nature. However, mutagenesis of BR has also been employed to introduce gold-binding capabilities via cysteine mutations (Brizzolara et al., 1997; Schranz et al., 2007), enhance the innate dipole moment (unpublished data), and overall photocycle speed of the protein (unpublished data). A general timeline for producing BR mutants is shown in Fig. 2.15.

2.15 Timeline for the genetic engineering of bacteriorhodopsin through site-directed, site-specific saturation, semi-random and random mutagenesis. The chart represents a single round or stage of mutagenesis, which is accomplished in approximately ten weeks. After ten weeks, preliminary characterization of mutants is carried out, and the best mutants for a specific trait are selected to serve as the template for subsequent rounds of mutagenesis in a process referred to as type I directed evolution.

Directed evolution (DE) provides a cost-effective and time-efficient method for genetically manipulating biological macromolecules, specifically BR, to serve in non-native environments (Wise et al., 2002; Hillebrecht et al., 2004; Hillebrecht et al., 2005). This process enhances a molecule toward a specific characteristic via repeated iterations of genetic mutation, screening and differential selection. Historically, DE has been used to modify properties of enzymes and biopolymers for industrial and pharmaceutical applications. In each case, a diversified genetic library was generated and appropriate screening methods were used to identify mutants with enhanced phenotypes that include substrate specificity, and chemical and thermal stability (Arnold, 1998; Kettling et al., 1999; Lin and Cornish, 2002; Jäckel et al., 2008). This approach allows for the deliberate evolution of individual populations of molecules toward a specific trait.

Protein redesign of BR by DE is accomplished through a combination of site-directed, site-specific saturation, semi-random and random mutagenesis. Site-directed mutagenesis (SDM) exchanges a single amino acid, typically a key residue, to alter the structure or function of the molecule. Semi-random mutagenesis (SRM) and random mutagenesis (RM) produce a large number of indiscriminate mutants through the use of doped primers or oligonucleotides. Selection of prospective SRM mutants is feasible because Halobacterium colonies are pigmented when the protein is functionally expressed. Site-specific saturation mutagenesis (SSSM), a combination of SDM and SRM, allows for the exploration of multiple amino acid substitutions via the saturation of a key residue at a specific locus on the bacterio-opsin (bop) gene.

In the first round of DE, region-specific SRM is used to generate a large number of mutants that are then screened with respect to a specific property. For BR, mutants are typically screened for altered photophysical properties, such as M state lifetime or Q state formation, using 96 well plates. Because the deliberate engineering of BR for device applications necessitates several rounds of mutagenesis to identify a mutant that outperforms the native protein, an in-house automated screening system was developed based on microgram characterization (Wise et al., 2002). Proteins exhibiting the desired trait are then grown up in sufficient quantities for further testing and are selected for subsequent rounds of mutagenesis via SDM, SSSM, SRM or RM. Through six-stages of DE, over 10 000 BR mutants have been screened and investigated for use in biomimetic devices. This process is illustrated for enhancing the Q state of BR in Fig. 2.16.

2.16 Six stages of directed evolution of bacteriorhodopsin to enhance Q state formation. (a) A 2D structure of bacteriorhodopsin that was generated using the program Protein 3.6 (Professor Robert R. Birge, Department of Chemistry and Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA). Bold residues represent the key sites responsible for the formation of the Q state. (b) Amino acids substitutions, which generated high amounts of Q, were used as the parents for the next generation of genetic progeny. After six rounds of mutagenesis, 1604 novel mutants were generated. The best mutants are currently being tested for application in 3D volumetric memories and holographic associative recall.

2.5 Conclusions and future trends

Biotechnology is now at a point where the macro- and the microstructures can be efficiently combined and improved upon with various techniques. The stable architecture of retinylidene proteins offers a template that provides significant biomimetic potential, not only because many of these proteins are very stable but also because of their unique photophysical properties. Bacteriorhodopsin has been one of the most successful targets because of the stability imparted by the semi-crystalline structural lattice. The recent applications of other retinylidene proteins, described in section 2.2.2, offer new and exciting advances in the field. Perhaps the greatest potential lies in the ability to manipulate retinylidene proteins via genetic engineering and protein redesign. Directed evolution provides the most significant potential when such can be implemented.

2.6 Acknowledgements

This work was supported in part by grants from the Defense Advanced Research Projects Agency (HR0011-05-1-0027), National Science Foundation (NSF-0829916), the National Institute of Health (GM-34548) and the Harold S. Schwenk Sr. Distinguished Chair in Chemistry.

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