Quarterly Reviews of Biophysics 24, 4 (1991), pp. 425-478

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Printed in Great Britain

Bacteriorhodopsin: a biological material for information processing DIETER OESTERHELT1, CHRISTOPH BRAUCHLE2 AND NORBERT HAMPP2 1 2

Max-Planck-Institute for Biochemistry, Martinsried, Germany Institute for Physical Chemistry, University of Munich, Germany

1. INTRODUCTION

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2. HALOBACTERIA AND THE PURPLE MEMBRANE

427

2.1 Structure and junction of bacteriorhodopsin 428 2.2 Generalization of the function of the retinal proteins 430 3. CHEMICAL AND GENETIC VARIATION OF THE PROPERTIES OF BACTERIORHODOPSIN AND RELATED RETINAL PROTEINS 431 4. TECHNICAL APPLICATIONS PROPOSED FOR BACTERIORHODOPSIN

432

4.1 Bacteriorhodopsin as a light-energy converter 436 4.2 Bacteriorhodopsin as a photochromic material 437 5. PROPERTIES OF BACTERIORHODOPSIN FILMS FOR OPTICAL INFORMATION PROCESSING 440

5.1 Recording and readout of information 443 5.2 Relation of absorption coefficient and refractive index 445 5.3 Holographic properties of bacteriorhodopsin films 447 6. BACTERIORHODOPSIN FILMS IN REAL-TIME INTERFEROMETR Y 7. BACTERIORHODOPSIN FILMS IN FOURIER OPTICS

450

455

7.1 Nonlinear optical filtering 456 7.2 Holographic pattern recognition 460 8. PHYSICAL AND OPTICAL DEMANDS FOR BACTERIORHODOPSIN FILMS IN TECHNICAL APPLICATIONS 9. ACKNOWLEDGEMENTS 10. REFERENCES

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468

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I. INTRODUCTION Technology which makes use of biological materials has advanced dramatically in the last few decades. Production of specific biochemicals by selected microbial strains, the use of enzymes for stereospecific biosynthesis of materials and gene

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Table i. Properties of the purple membrane (PM) and bacteriorhodopsin (BR) Purple membrane • 2-D, hexagonal crystal of uniaxially oriented trimeric BR • Contains x 10 molecules lipid per BR (buoyant density: i, 18 g/cm 3 ) • PM patches may be round (then small) or irregular (then larger) with diameters up to 5 /tm and a thickness of 5 nm • in vitro enlargement by fusion is possible • Deposition by electrophoresis or centrifugation to oriented films • Mixing with water-soluble polymers (poly-ethylene-glycol, poly-vinyl-alcohol, gelatine) for fabrication of mechanically stable films. • Films with adjustable water content and refractive index (nPM = 1-4-1 -5) • Stable under the following conditions Constant illumination (sunlight for years) Air (oxygen) in presence of light Temperatures over 80° Acidity lower than pH o Alkalinity above pH 12 High ionic strength Incorporation into polymers Presence of most proteases • Drying preserves colour and photochemical activity Bacteriorhodopsin • Reversible changes of the a\\-trans^ 13-czs configuration under standard conditions (pH 3;5~9°) • Formation of a g-cis form in deionized purple membrane and at acidic pH • Reversible protonation of the Schiff base • Quantum efficiency of the primary photoreaction ^>B_j j> 0-64 • Temperature dependent stabilization of intermediates (M, N, O) • Sensitive to most organic solvents • Orientation of the retinal chromophore Cyclohexene ring perpendicular to membrane plane long axis of retinal chromophore tilted 21° towards extracellular side • No refractory period of the photochemical cycle

technological production of biologically important macromolecules are a few examples of these developments. All these procedures involve the use of biological catalysts of metabolic pathways to some extent. Attempts to exploit biological energy converters as tools for hydrogen or ATP production through the biotechnology of solar energy conversion have been less successful but are areas of current research. A new field of application for biological materials is their use in information technology where strategies employing small structures ('nanostructures') down to the molecular scale ('molecular electronics') and the use of optics present modern trends. Although the so-called optical computer is still a speculative project regardless of the material planned for its construction, the use of photochromic materials, and the advantages offered by non-linear optical

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materials, for information storage and processing can be envisaged as having great potential. Among the biological materials proposed for such projects bacteriorhodopsin stands out as being one with particular advantages. It is available in virtually unlimited amounts and has thermo- and photochemical properties ideally suited for use in technical processes. The surprising stability of this material and the physicochemical features of the purple membrane enable us to engineer various technical systems such as bulky but optically homogeneous materials or oriented ultrathin films. In addition, genetic engineering of bacteriorhodopsin has become possible recently. Thus for the first time variants of bacteriorhodopsin exist and mark the strategy for the construction of artificial molecules. One example exists already where the optical properties of the molecule have been modified and improved by gene technological methods. This review aims to introduce bacteriorhodopsin as one of the most promising biological materials for application in information storage and processing but also describes the attempts for its use in energy conversion. 2. HALOBACTERIA AND THE PURPLE MEMBRANE

Bacteriorhodopsin (BR) mediates photosynthesis in Halobacteria, the extreme halophilic branch of the archaebacterial kingdom (for review see Oesterhelt, 1989). The molecule, however, does not contain chlorophyll which is the normally typical pigment in photosynthesis, but rather is a member of the retinal protein family of these organisms and functions as an outwardly directed proton pump located in the bacterial cell membrane (Oesterhelt & Tittor, 1989). Similar molecules act as inwardly driven chloride pumps [halorhodopsins (HRs), Lanyi, 1990] or sensory pigments for detection of the light quality of the environment [sensory rhodopsins (SRs), Spudich & Bogomolni, 1988]. These three functions enable halobacteria to find the optimal conditions for phototrophic growth in an otherwise hostile environment of salt and high temperature with light as the only energy source. Under conditions of extreme induction the intrinsic membrane protein BR can cover up to 80 % of the cell surface of a halobacterial cell. Upon exposure to pure water the cells disintegrate and release membrane fragments containing BR which, due to the colour of BR, are named purple membranes. Methods have been developed to isolate the purple membrane, which consists exclusively of BR and lipids, by a filtration technique without the requirement of centrifugation (Neumann & Leigeber, 1989). From a 100 1 fermenter culture about 6 g of purple membranes can be isolated which have a surface of 1000 m2. Properties of the purple membrane are summarized in Table 1. Apart from its astonishing chemical stability, BR has the remarkable feature of forming a hexagonal crystalline array of trimers with lipid filling in the gaps. This is one of the few naturally occurring 2D protein crystals and explains, in part, the stability of BR which is lost upon solubilization of the membrane by detergents. The stability of the purple membrane against temperature, drying out, salt, extreme pH values and organic polymers is unique for a biological material. Even more surprising is the fact that

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(a)

(6)

Fig. i. Topography of the helices of BR (a) and scheme of the proton translocation through the protein (6). The retinal chromophore is bound to helix G via lysine (Lys) at position 216 (K216). The aspartic acid (Asp) residues 85 and 96 (D85, D96) act as proton donor and acceptor during the proton translocation. The light-induced (hv) proton transport is directed from the cytoplasmatic (CP) to the extracellular (EC) side of the membrane.

under all conditions, including polymerization thermoreversible photochemistry persists.

into

silicone

rubber,

2.1 Structure and function of bacteriorhodopsin All halobacterial retinal proteins belong to the seven-transmembrane helix family of membrane proteins (Henderson & Schertler, 1990). Eucaryotic members are represented by /?-adrenergic receptors and visual pigments. The only structure of a protein of this topography which has been suggested as a model at near atomic resolution is the BR (Henderson et al. 1990). The chromoprotein has a molecular weight of 26 kDa with the retinal moiety bound to lysine residue 216 in the form of a protonated Schiff base. The seven-transmembrane helices (A-G) are arranged in a circular manner and a transmembrane pore is formed mainly by helices B, C, F and G (Fig. 1). The Schiff base is located approximately halfway through the cross-section of the total membrane span of 48 A and separates an extracellular (EC) half channel from a cytoplasmic (CP) half channel. The cyclohexene ring is oriented perpendicular to the membrane plane and the long axis of the chromophore is tilted 21 0 towards the EC channel (Ebrey et al. 1977; Heyn et al. 1977; Lin & Mathies 1989). Ion translocation is believed to proceed through this pore via the Schiff base itself. Indeed at least four water molecules could be located in this area of the molecule as deduced from neutron diffraction (Papadopoulos et al. 1990). The retinal itself extends from its attachment point on helix G all the way across the interhelical space with a tilt of about 21° with respect to the plane of the membrane (Hauss et al. 1990). The molecule in its binding pocket is closely packed with tryptophan residues and the cyclohexene ring moiety touches helices D and E. A complex anion formed by the surrounding amino-acid side chains provides the

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Fig. 2. Photochemical and thermal conversions of BR. The intermediates are abbreviated by single letters. B represents the initial light-adapted state and D stands for dark-adapted BR. Indices give the absorption maxima of the states. M1 and M" both absorb at approximately 410 nm (Varo & Lanyi, 1990).

counterionic charge for the positively charged nitrogen of the Schiff base and the distance between these charges controls the colour of the chromophore (de Groot et al. 1989; Blatz et al. 1972; Schulten et al. 1980; Tavan et al. 1985; GroBjean & Tavan, 1988). In the dark, the chromophore occurs as a mixture of an alltrans, 15-anti and a i2-cis,i^-syn configuration (Smith et al. 1984). Both forms are photoactive but only the trans, anti-configuration mediates proton translocation by its photochemistry. Under constant illumination it accumulates to nearly 100%, absorbing maximally at 570 nm. Absorption of a photon by this trans,anti-configuration causes isomerization around the 13,14 double bond within 0-45 ps with a quantum yield of about C64 (Dobler et al. 1988; Mathies et al. 1988; Tittor & Oesterhelt, 1990; Govindjee et al. 1990; Xie, 1990; Birge, 1990 a). The first photochemical product J then thermally relaxes via intermediates K and L to release a proton from the Schiff base via residue 85, which is an aspartic acid (D85), into the EC channel of the structure (Butt et al. 1989; Marinetti et al. 1989; Subramaniam et al. 1990). This causes a dramatic blue shift of the chromophores absorption to the maximum of 410 nm in the M intermediate. Another aspartic acid, residue 96 (D96), participates in the re-uptake process of a proton through the CP channel (Holz et al. 1989; Tittor et al. 1989; Miller & Oesterhelt, 1990). The intermediate N, then formed, is able to re-isomerize around the 13,14 double bond and subsequently the initial state is regenerated by re-establishment of the original protonation states of all side chains. Fig. 2 summarizes the spectroscopically discernible intermediate states of the catalytic cycle and puts emphasis on the two M states

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which have been subject to recent investigations (Varo & Lanyi, 1990, 1991). In M 1 the Schiff base must have a low pK and the proton is released to the extracellular side whereas in the M11 state it has a high pK and accepts a proton from the cytoplasmic side. Another property of the M intermediate, which is particularly important for the speed of optical processing in BR, is noteworthy and is described in section 6.1. If M captures a photon, i.e. blue light is present in addition to the yellow light necessary for M formation, M is photochemically, not thermally, reconverted to the initial state (Oesterhelt et al. 1975; Ormos 1980). In this two-photon cycle, it is not the thermal rate of M decay (about 5 ms) but its thermal formation from the L intermediate (about 50 fis) which limits the write and erase cycles (see below). In agreement with its expected role as proton acceptor in the first half of the catalytic cycle D85 has been found to be deprotonated in the active BR pump molecule. The presumed proton donor D96 on the other hand is protonated under the same conditions as found by FTIR-measurements (Gerwert et al. 1989; for reviews on vibrational spectroscopy see: Kitagawa & Maeda, 1989; Siebert, 1989). The details of the structure and the mechanism of function have been reviewed recently and the reader is referred to Tittor (1991) and Trissl (1990).

2.2

Generalization of the function of the retinal proteins

The primary structure of the polypeptide chains of the related proteins HR and SR from Halobacterium halobium, Halobacterium sp. GRB and other halobacterial strains are also known (Soppa & Oesterhelt, 1989; Soppa et al. 1989; Blanck & Oesterhelt, 1987; Blanck et al. 1989; Lanyi et al. 1990; Uegaki et al. 1991). They all are homologous structures. Amino-acid residues involved in retinal binding in the BR molecule are highly conserved in all of these proteins. Another group of residues which are involved in the formation of the ion pore are strictly conserved within each of the functionally different families, but differ depending on the respective function as proton or chloride pumps or as sensors. In all three cases the molecules isomerize around the 13,14 double bond and can return thermally back to the trans state. This reversible transition is linked to reversible deprotonation of the Schiff base characteristically in BRs. In SR a similar process occurs but an intermediate is formed which absorbs at 373 nm and has a half-time of decay of 700 ms instead of 5 ms in BR. All SRs analysed so far lack D96 as an internal proton donor in the CP channel which makes the BR molecules function as fast and pH-independent proton pumps. All SRs, however, have D85 as proton acceptor in the EC channel and this explains the observed rise of the SR 373 intermediate in the [is range. A characteristic feature of the catalytic cycle of HR is that no deprotonation occurs but the molecule cycles at the speed of BR through the 13-as state without losing its charge at the Schiff base nitrogen. Connected to this charge movement by trans/cis isomerization is the translocation of the chloride ion (Oesterhelt et al. 1986; Oesterhelt & Tittor, 1990). Clearly, one would not expect HR to carry either

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D85 or D96 on its polypeptide chain and indeed none of the HRs so far analysed disobey this rule. Interestingly, in one in several thousand cycles HR spontaneously deprotonates and is trapped in the so-called HR-410 state which only decays by reprotonation having a half-life of minutes. Blue light absorption or the addition of the inorganic anion azide (and some others) speeds up this process into the ms range (Hegemann et al. 1985 a). Blue light thus acts as an activator for the green light-driven, but at the same time green light-inhibited, chloride pump (Hegemann et al. 1985 ft). Azide, on the other hand, acts as a protonophore for the CP channel in HR. Azide has the same property with BR which is best exhibited in mutants lacking aspartic acid 96 (Tittor et al. 1989). 3. CHEMICAL AND GENETIC VARIATION OF THE PROPERTIES OF BACTERIORHODOPSIN AND RELATED RETINAL PROTEINS

BR and related molecules can be modified in their properties by altering four parameters: 1. by the physicochemical conditions of the medium such as temperature, pH, salt and water activity; 2. by the ion substrate concentration which is pH for BR and the chloride (halide) concentration for HR; 3. by replacement of retinal in the binding site with retinal analogue structures; 4. by genetic modification of the primary structure of the polypeptides. The main properties of the molecules which can be varied are the colour of the initial state and of the intermediates during the photochemical cycle and the stability, i.e. the half-time of decay, of such intermediates. In this regard secondary photochemistry, i.e. a sequence of two photochemical processes, can play an important role. One example of the concerted action of yellow and blue light on wild-type (WT) BR was already described above. A few examples should illustrate the statements made above. (1) Temperature and pH changes influence the lifetime of the intermediates (e.g. M, N, O) and thus change their distribution under photostationary conditions. Drying of purple membrane films will cause mainly photoactivity of the 13-m, 15 syn (dark-adapted) state (Varo et al. 1991; Varo & Lanyi, 1991). (2) pH values below 2 cause protonation of D85, i.e. blockage of the proton acceptor and a blue colour. As a consequence no M intermediate forms and a photochemistry, possibly even ion translocation activity, similar to HR is created (Keszthelyi et al. 1990). Similarly changes of the substrate concentration, e.g. the absence of chloride, causes a change of the initial state properties of HR and a corresponding change in its photochemistry. In both cases a side reaction of low quantum yield leads to a g-cis chromophore species with an extremely long lifetime (Maeda et al. 1980; Fischer et al. 1981; Zimanyi & Lanyi, 1987). (3) Retinal in the binding site of BR has been replaced by literally hundreds of different chemical structures and the modified properties again concern colour, 16

QRB 24

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photochemical behaviour and proton translocation activity. Only a few examples should demonstrate the almost inexhaustible possibilities of variation. An analogue called 'phenylretinal' (Rolling et al. 1984; Polland et al. 1984) prevents photochemistry in BR for steric reasons but provides, in principle, an 'optical switch' material in the 10 ps range. Azulene derivatives extend the absorption range of BR to the near infrared (IR) (Asato et al. 1990) and 13-trifluoromethylretinal derivatives bring the modified BR into the range of diode laser wavelengths (Gartner et al. 1981). Depending on the number of conjugated double bonds, material of almost any absorption characteristic in the visible and near-IR can be created in principle and similar ideas apply for the long-lived intermediate states important for photostationary conditions, i.e. constant illumination. The lifetime of some photoproducts increases from 5 ms to relative infinity as demonstrated by 13-methoxyretinal in BR, where the photoproduct lives on a ms time scale practically for ever (Gartner & Oesterhelt, 1988). In summary, a plethora of chemical compounds can be used to modify the properties of BRs and other retinal proteins. (4) Genetic methods to facilitate manipulation of BR and other halobacterial retinal proteins have been applicable for some time, with the recent establishment of a halobacterial transformation system being a key event (Lam & Doolittle, 1989). Random and site-specific mutagenesis followed by either selection or serendipitous choice and characterization of the mutants enable innumerable changes of the primary structure of BR and its properties. A single example of a mutated BR structure will exemplify the success of the genetic approach. Mutant 326, selected for the inability to mediate phototrophic growth, carries an asparagine instead of aspartic acid in position 96. Due to the lack of the internal proton donor this causes not only a drastic increase of the lifetime of the M intermediate but also its strict dependence on the pH in the medium (Miller & Oesterhelt, 1990), since reprotonation then depends on bulk pH. For possible technical applications of BR the possibility to vary one of the properties of the protein over several orders of magnitude, by adjusting a few physico-chemical parameters of the medium, is of high value. For example, when information is recorded with light and the light-induced population distribution is used for its storage, the increase of the M lifetime is useful. This is possible with mutant 326. In other, e.g. photoelectric applications, different parameters adjustable by exchanging other amino acids might become important. For all variations, one must realize that BR has an almost unique chemical and photochemical stability due to its arrangement in the lipid bilayer of the purple membrane. Although the variations mentioned are powerful tools for the design of new materials, the possible destabilization of the protein resulting in a drastically decreased durability of the material must be considered in every case.

4. TECHNICAL APPLICATIONS PROPOSED FOR BACTERIORHODOPSIN For a possible technical application of BR exclusively the purple membranebound state of BR was investigated so far and therefore is used synonymously throughout this article. As described in the previous sections only the purple

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Table 2. Proposed technical applications of bacteriorhodopsin Proton pumping and photoelectricity

Photochromism

ATP generation in reactors Desalination of seawater Conversion of sunlight to electricity Ultrafast light detection Chemo- and biosensors Information storage Storage devices and recording processes Holographic storage Information processing Signal conditioning Real-time interferometry Spatial light modulators and optical filtering Phase conjugation Associative memories Pattern recognition Neural networks Related fields 2nd harmonic generation Bistability Light switching

(1)

(2)

(3) (4) (5) (6) (7) (8) (9) (10)

(11) (12)

(13) (14) (i5)

(16) (17)

(1) Inatomi (1984); Maximychev & Chamorovskii (1988). (2) Oesterhelt (1976). (3) Drachev et al. (1976); Eisenbach et al. (1977); Bamberg et al. (1979); Seta et al. (1980); Singh et al. (19800, 6); Singh & Caplan (1980c); Vard & Keszthelyi (1983); Maximychev et al. (1984); Oagawa (1985); Keszthelyi et al. (19906). (4) Trissl (1987); Trissl et al. (1989); Katsura et al. (1989). (5) Vsevolodov & Ivanitskii (1985 a); Mukaihata (1986); Fowler & Devonshire (1991); Lee et al. (1991).

(6) Isoda & Daimon (1984); Isoda (1984 a, b); Arai et al. (1986 a, b)\ Lee et al. (1989); Birge (1989); Margalit & Yu (1990). (7) Bunkin et al. (1981); Vsevolodov & Poltoratsky (19856); Vsevolodov et al. (1986); Bazhenov et al. (1987); Hampp et al. (1988, 1989, 1990a, 19916); Zeisel & Hampp (1991). (8) Barmenkov et al. (1987); Abdulaev et al. (1988); Barmenkov et al. (1988). (9) Razumov et al. (1989); Hampp et al. (1990c, d); Renner & Hampp (1991). (10) Hampp et al. (1990 c); Korchemskaya et al. (1990); Birge et al. (1990 a1); Thoma et al. (1991). (11) Korchemskaya et al. (1987); Werner et al. (1990); Hampp & Brauchle (1990/). (12) Birge et al. (1990a1). (13) Hampp et al. (19906, e, 1991a). (14) Mobarry & Lewis (1986); Takei et al. (1991); Haronian & Lewis (1991). (15) Aktsypetrov et al. (1987); Huang & Lewis (1989); Huang et al. (1989); Birge et al. (1990 c). (16) Vsevolodov et al. (1981); Bazhenov et al. (1989); Vsevolodov et al. (1989). (17) Van Brunt (1985); Hikima et al. (1985); Karube (1986); Hong (1986); Inoue (1987 a); Rayfield (1989).

16-2

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Natural system

BR wild-type

Optical medium

Engineering Diversification

\

fiR

Reversibility Thermal Photochemical

Stability Thermal Chemical Photochemical

Spectral range VIS & near IR

RR variants BR variants Genetic engineering

Data

„

U gg hh tt U

\ y

recordin

S

m oo dd uu ll aa tt oo rr ss m

Nonlinear filtering Interferometry Phase conjugation Pattern recognition Neural networks Associative etc memory

Availability Unlimited Fig. 3. A new direction in material science - BR as a model. The attractive properties of BR wild-type are shown in the left column and possible optical applications with differing physical demands are listed in the right column. Engineering of BR and purple membrane suspensions is necessary to produce materials suitable for these applications. BR films with the required optical quality (optical flatness, homogeneity, scattering, aperture, etc.) and containing a wide variety of BR variants (improved light sensitivity, spectral range adapted to cheap, small and reliable light sources, stabilization of intermediates at room temperature, colour-fastness) are technically feasible.

membrane form exhibits the thermal and photochemical stability which is necessary for technical applications. All ideas about application of BR reported are based on one of the following basic properties of BR. 1. 2. 3. 4. 5.

BR is a light-driven proton pump; BR generates a protomotive force of about 300 mV across the membrane; BR is a chromophoric protein; the initial photochemical conversion of BR (B -> J) is very fast; the absorption spectrum of BR and its photochemical characteristics are sensitive to the surrounding medium.

The excellent stability of this membrane protein and the continuously increasing knowledge of its molecular properties, its structure and its function (Kouyama et al. 1988), have raised hopes, even very early after its discovery, for technical

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applications of BR and many detailed proposals have been published over the years (see Table 2). However, no prototype of any device based on BR could be presented and no commercial product exists so far where a functional component is made from BR. This confirms the unwritten rule that in technical applications the use of synthetic materials is preferred. They can be made in reproducible form and are usually available in unlimited amounts. In addition they can be engineered and thereby their properties can be adapted to specific applications. Natural materials, although optimized for their function, are normally lacking in this possibility. BR, however, is an exception. Retinal or retinal analogue structures can be added to the growth medium of halobacteria in vivo or to chromophore-free membranes in vitro. In addition, variants of the polypeptide chain of BR can be generated by conventional mutagenesis and isolated by a specific selection method (Oesterhelt & Krippahl, 1983; Soppa & Oesterhelt, 1989; Soppa et al. 1989). Sitespecific mutagenesis of the BR gene and expression in E. coli (Nassal et al. 1987; Holz et al. 1989) provides a free choice in the modification of BR. However, the thermally stable purple membrane form of BR could only be produced after the establishment of a halobacterial transformation system (Lam & Doolittle, 1989) allowing creation of new strains at will. This opens new avenues in material science. Besides the possibility to engineer BR, naturally occurring BR itself has attractive properties. Worthy of mention are its excellent stability towards thermal, chemical and photochemical degradation, a good photosensitivity ( 0 B _ j ^ 0.64, eB(57o nm) = 63000 1 mol"1 cm"1) and the combination of thermal and photochemical pathways in its photocycle which allow the construction of purely light-controlled systems. Its spectral range covers almost the whole visible region. Due to the ease of cultivation of halobacteria isolation of purple membranes on a technical scale is feasible. The basic properties of BR can, in principle, be used directly in many optical applications (see Fig. 3, right row). The goal of genetic engineering is to meet the specific physical demands of different applications more closely, since numerous different BR variants with modified optical properties can be generated. In this section proposals for technical applications of BR shall be briefly listed (see Table 2) to give an impression of the wide variety considered to be technically possible. Since the use of BR - mainly focusing to wild-type BR - was reviewed by several authors (e.g. Hong, 1986; Vsevolodov et al. 1989; Bazhenov et al. 1989; Birge, 19906) we shall give only a short overview of the basic principles. The use of engineered BRs (Hampp & Brauchle, 1990/; Brauchle et al. 1991) will be discussed in more detail in sections 6, 7 and 8, in particular focusing on optical information processing. The physical demands required of the BR materials shall be derived from a few selected examples and the performance of BR variants in these applications will be reported.

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4.1 Bacteriorhodopsin as a light-energy converter In the later 1970s and early 1980s the interest focused on BR as a light-driven proton pump and as a system for the conversion of sunlight into chemical or electrical energy. The light-driven proton translocation through the protein (Kouyama & Nasuda-Kouyama, 1989) could be incorporated as a functional part in artificial systems. For example, thin layers or foils containing oriented BR molecules act, under illumination, as large area proton pumps and generate a light-induced vectorial proton transport through the artificial membrane. Such systems have proven to be functional under laboratory conditions with small-sized and lowcapacitive membranes (Bamberg et al. 1979; Singh et al. 1980 a). A test under realistic environmental conditions has not been reported. The performance of such systems is dominated by two factors. First, the degree of orientation of the PM patches in the film, and second, the apparent quantum efficiency for the proton translocation, i.e. how many photons are needed to transport a single proton through the artificial membrane. Theoretically only a single layer of PM patches and about 1 photon per proton are required. However, it is very difficult to create a high ratio of light-induced proton transport capacity and diffusional impermeability. This can so far only be achieved by bulky membranes which have a lower apparent quantum efficiency. This is reason enough why the use of BR for the conversion of sunlight into electricity has failed. Additional problems arise from the interface between proton pumping BR and conventional electron storage devices, e.g. batteries. Furthermore, the technique and the efficiency of silicon-based solar cells (Pleskov, 1990) have reached levels where alternatives on the basis of BR cannot and are unlikely ever to be able to compete. The efficiency of standard solar cells ranges up to 14% compared with about 1 % calculated for BR-based systems (Singh & Caplan, 1980 c). In principle, the proton gradient generated between two compartments by the action of BR can be used for ATP regeneration from ADP in a reactor where the same processes take place as in the halobacterial cell. The gradient generated by a BR-based macroscopic proton-pumping membrane is utilized by an ATP synthase bound to a second membrane (Inatomi, 1984; Maximychev & Chamorovskii, 1988). The low overall efficiency of such systems would be compensated by the price of the produced ATP. The main drawback of this process is that waste ADP must be collected and transported back to the ADP/ATP-recycling reactor. The resulting costs for transport and handling presumably will restrict the use of such a reactor for stationary systems, e.g. in fermentation plants. An interesting approach is to use the proton-pumping activity of BR for the desalination of seawater as proposed by Oesterhelt (1976). Due to the increasing need for drinking water in coastal and desert areas this might be of future economical importance. For local needs, the use of BR and also HR might be a reasonable addition to conventional methods. This research direction has not been

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extensively investigated. The price of BR is still too high for the construction of full-scale systems and many technological problems are still not solved like the stability and uniformity of large-sized foils with immobilized and highly oriented BR. The very fast initial steps in the BR photocycle are the basis for light-detection systems with BR as an active element. Trissl et al. (1989) presented a photodetection system made from an oriented BR sample coupled to the end of a coaxial cable. The time limit in the rise-time was determined to be approximately 20 ps. This is somewhat slower than silicon photodetectors which reach rise-times as low as 1 ps when optimally coupled to the monitoring device. The use of BR in biosensors was also proposed. The basis for such sensors is the modification of the BR photocycle and/or its absorption spectrum by an organic compound, e.g. anaesthetics (Henry et al. 1988), by the addition of organic dyes (Mukaihata, 1986) or by the presence of organic solvents (Uehara et al. 1990; Mitaku et al. 1988). This type of biosensor may be quite sensitive but the selectivity is rather poor. Therefore no technical use seems imminent.

4.2 Bacteriorhodopsin as a photochromic material The outline of structure, function and physicochemical properties of BR in sections 2 and 3 can be focused in a statement about the essential features of BR as a photochrome (material with light-inducible reversible colour changes) with technical potential. The protein part of BR guarantees a stereoselective and thermoreversible photoisomerization of retinal with high quantum yield and without side reactions. This makes BR a material more light stable than most known organic photochromes. Even if side reactions occur under certain conditions they do not lead to destruction but rather to photochemistries on different time scales in the same molecule. The primary reaction under standard conditions occurs on a very short time scale, subpicoseconds, and with a change in colour. Further colour changes occur in the ps, /is, lower ms and in the upper ms range for the dominant change to yellow (M intermediate). The initial state, the purple B state, can be regenerated thermally from the M state but also photochemically from all other intermediate states. The M->B photochemical transition has already practical importance and will be described in detail as M-type holographic recording below. Mixtures of states or mixtures of WT and mutant or chemically modified proteins can be used for parallel processing of optical information. The colours and their switching times in BR can be manipulated over a wide range by use of retinal analogues, gene technological manipulation of the protein part and the change of the environmental parameters. The photochromic properties, the reversibility and the speed of the BR system suggest a technical application particularly in fields where a combination of several properties creates optimal features. Optical data storage and optical information

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processing are such examples and a few years ago several groups started to investigate different approaches and applications (see Table 2). The various optical techniques (Fig. 3) have specific demands on the media used for recording and/or processing of information. They differ with respect to the recording and erasure sensitivity, storage time, resolution, wavelength regime, response time and signal-to-noise (S/N) ratio and can further be classified into devices where only the light-induced absorption changes and the resulting contrast ratio (e.g. in point storage devices) or the light-induced changes of both, the absorption coefficient and the refractive index are utilized, e.g. in holography. Magneto-optical recording (Chen, 1974) is the dominating technique today for optical data recording (Emmelius et al. 1989; Ravich, 1989). Mass storage devices are commercially available from several companies (e.g. Sony) at affordable prices. In magneto-optical point storage devices, which allow diffraction limited recording, the combined action of a magnetic field and an optical induced phase transition is used for recording and erasure. For readout only light is needed and therefore the information is not degraded or even destroyed by the readout process. Such a 'gating' mechanism is lacking for photochromic materials and also for BR. Furthermore it is not possible to stabilize at room temperature at least two different BR states (e.g. B and M states) for the required periods of 10 years. Cooling to lower temperatures, e.g. —40 °C (Birge, 1989) allows the combination of the fast photoresponse of BR (B -»• J) and a virtually unlimited storage time (T M ->OO). Even in the case that the necessary technical efforts are neglected the performance, i.e. the storage density (bits/cm2) and the storage time (10 years) of BR-based point storage devices cannot be improved beyond the performance of magneto-optical media. The photoactivity of intermediates of the BR photocycle (Balashov & Litvin, 1981) may be employed for a non-destructive readout process (read without erase) for information recorded in BR films. Two light beams of wavelengths Ax and A2 are adjusted with respect to their intensities / x and I2 so that the forward reaction B->K induced by A1 and the backward reaction K -* B compensate exactly and no net change of population distribution occurs during reading. This simultaneous use of A1 and A2 was first suggested by Birge (19906) and extended further by development of a delayed light pulse system by Haronian & Lewis (1991). This technique is restricted to storage devices for digital information but is not reliable enough for analogue information devices. The photoactivity of the K intermediate of BR might be utilized at lower temperatures also for different purposes, e.g. for ps switching (B±^K). Replacement of the retinal chromophore by phenylretinal (Polland et al. 1984) provides a material which allows ultrafast switching at room temperature or information recording in the excited state. A wide range of recording processes has been described and/or patented for BR (see Table 2). A type of 'chemically gated' recording was presented by a combination of BR and the protease chymotrypsin which is sensitive to UV (Arai et al. 1986 a). It was claimed that in positions where the film is irradiated with UV the enzyme is activated and then inactivates BR. In the areas of functionally active

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BR, light exposure should lead to the release of protons from BR and the formation of a blue picture by the added pH indicator, bromothymol blue. A further claim concerned a mixture of BR types with three different retinal analogues which was proposed for colour recording and readout processes (Arai et al. 19866). BR as a photostable but ultrafast photochromic system at low temperatures (liquid helium) in photochemical holeburning (Wild et al. 1990) was successfully demonstrated but is not applicable for information storage since the halfwidth of its homogenous absorption line is almost as broad as the band at room temperature (Lee et al. 1989). BR therefore allowed the storage of only minor amounts of information in its absorption band. Permanent information recording with BR at room temperature is possible by the addition of hydroxylamine. In the illuminated areas BR is sensitive to hydroxylamine and irreversibly forms retinaloxime which absorbs around 360 nm (Oesterhelt et al. 1974). Under these conditions BR would be functionally equivalent to silver halide films, but orders of magnitudes would be less sensitive. A more promising area for use of BR is transient information storage which is needed in optical information processing. In dynamic applications BR can be used as a medium to catalyse the interaction of two light waves. The most interesting and realistic suggestions in this direction are listed below. The application of BR as an optical data recorder with short access time, fast response and with short erase times was proposed (Margalit & Yu, 1990) as a buffer-like, short-term storage medium for the recording and preprocessing of data from synthetic aperture radar (SAR) systems. The electronic equivalent of this function is called 'serial-in parallel-out buffer'. An experimentally verified application is temporal low-pass filtering of optical signals for signal conditioning. An optical signal with intensity noise is optically low-pass filtered when transmitted through a BR film. Barmenkov et al. (1987) see the advantage of BR in this application that a time domain (1—100 ms) which is not easily reachable with alternative materials such as photorefractive media of high sensitivity are accessible by dynamic holography in BR films (e.g. Barmenkov et al. 1987). Similar response times in the /is range are shown by thermal gratings which, however, need high light intensities. Real-time interferometry with BR (Razumov et al. 1989; Hampp et al. 1990^; Renner & Hampp, 1991) is based on the time-integrating properties of BR films which are determined by the light-dependent B -> M and M-lifetime-dependent M -* B reactions. In this technique detection of vibrational modes by time integration of holographic images is performed. A more detailed description of this method and the use of BR films is given in section 6. Spatial light modulators (SLMs) formed with BR are based on two different principles. The first one uses the light-intensity ratio-controlled absorption of BR. A BR film can be used to control light of a first wavelength which induces the photoreaction B -> M with a second light wave inducing M ->B. The local intensity ratio determines to what extent the two wavelengths are absorbed in the BR film (Thoma et al. 1991). The second principle uses the photoelectric

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properties of oriented BR films. The light-induced translocation of charge, i.e. protons, can be enhanced or suppressed by external electric fields (Tsuji & Hess, 1987). Spatial control in such an electrically addressed SLM needs a pixelated electrode system. The resolution of these devices is limited by the structural sizes achievable for wiring in such systems (/tra range). Therefore, the molecular resolution of BR cannot be used in these SLMs and since many reliable pixelated devices based on liquid crystals or deformable mirrors have been developed (Efron, 1990) BR does not seem to have a specific advantage. However, detailed experimental studies are necessary for a more critical evaluation of BR in SLM technology, which is the key to efficient optical processing. Optical phase conjugation (Zeldovich & Shkunov, 1979; Martin et al. 1980) with BR (Korchemskaya et al. 1987; Hampp & Brauchle, 1990/; Werner et al. 1990) is based on volume holograms. The efficiency of the system is, however not very attractive, but its sensitivity and its quality are interesting. BR can be used for the restoration of the polarization state in the conjugated wave as first described by Korchemskaya et al. (1987). Associative memories and neural networks also involve volume-type holograms. Proposals for BR (Birge et al. 1990c/) were made on the basis of a setup described by Peak & Psaltis (1987). Optical pattern recognition with BR is still in its infancy (see also section 7.2). It seems a promising application taking advantage of several specific properties of BR (Hampp et al. 19906, 1991 a). Takei et al. (1991) and Haronian & Lewis (1991) introduced the photoelectric properties of BR for the design of neural networks. Oriented BR films producing ' photoactivated diode junctions' can be the basis of such networks. The difference to electronic systems is that reconfiguration of the interconnections (reprogramming) can be done by light. Other fields like second harmonic generation (SHG), optical bistability or light switching have not yet claimed to be technically feasible (Aktsypetrov et al. 1987; Huang et al. 1989) and the more advanced applications of BR such as in ' biochips' and ' biocomputing' are still futuristic (Van Brunt, 1985; Hikima et al. 1985; Hong, 1986; Karube, 1986; Inoue, 1987 a). 5. PROPERTIES OF BACTERIORHODOPSIN FILMS FOR OPTICAL INFORMATION PROCESSING

For a detailed discussion of the ready-to-use BR film we shall restrict ourselves to the area of transmission holograms and not treat reflection holograms. Holography is a method which allows recording of the amplitude and phase of a light wave reflected from, or transmitted through, an object. With a setup as shown in Fig. 4a a transmission hologram can be recorded. The beam from the laser is split (BS) and expanded by lenses L. One beam, the so-called object beam O, is transmitted through the object, e.g. a slide, and thereby it is modulated in phase and amplitude. In the recording plane where the holographic medium HM is installed the object beam O is overlapped with the reference beam R. The amplitude and phase information carried on the object wave is coded by interference with the coherent reference wave as an intensity pattern, which is recorded in the holographic medium and thereby the hologram is formed. The

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(a) Recording

object

M

(b) Readout Holographic grating

(c) Simultaneous recording and readout Laser readout Laser

record

HO'

O

BR film

Object Fig. 4. Principle of holographic recording and readout, (a) The two coherent waves, object wave O and reference wave R, are overlapped and an intensity grating which codes the amplitude and the phase of the object wave is obtained. This pattern is recorded by the holographic medium and a grating with the fringe spacing G is formed. (b) Illumination of the recorded hologram with the reference wave results in a diffracted wave HO which is identical to the original object wave in phase, amplitude and direction, (c) Recording and readout process are performed in parallel in transient holography with reversible media like BR films. For the readout a reference wave R' which usually has a different wavelength than the recording reference wave R is used.

most simple hologram which is created if no object is placed in the setup is that of two plane waves; a periodic intensity grating I(x) of bright (white) and dark (black) areas is obtained. This can be mathematically described by

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where / B and Io are the intensities of the reference and the object wave (R and O) and V=2is the contrast of the pattern which is V = i for 7R = 7O and the fringe spacing G-

A

where A is the wavelength of the recording beams and 6 is the half-angle between object and reference beam. The photochemical process leads to an intensitydependent formation of photoproduct in the bright areas and leaves the holographic medium unchanged in the dark areas. This results in a periodic modulation of the refractive index .

277X

n(x) =«„ + «! cos—, and of the absorption coefficient .

27TX

a(x) = «„ + «! cos—, which are for low exposures a linear function of the incident intensity pattern. The values n0 and a0 represent the average refractive index and the average absorption coefficient in the grating and nx and a1 give the modulation amplitudes of both parameters (Brauchle & Burland, 1983). Diffraction of a light wave which is identical to the reference wave used during recording results in the reconstruction of the original object wave both in phase and in amplitude during the readout process. The relation of readout intensity 7B and the obtained diffracted intensity 7D is called diffraction efficiency rj and is given for a plane wave transmission hologram of thickness d by the relation -2aod\ cos 6 /' which was derived by Kogelnick (1969). Reconstruction of the recorded hologram can be done with a reduced setup of Fig. \a which is shown in Fig. 46. Only the reference wave R illuminates the hologram. The diffracted wave HO is a complete reconstruction of the original object wave in phase, amplitude and direction. For dynamic materials like BR films both processes, recording and reconstruction, are done in parallel (Fig. 4c). Since the readout wave usually is of different wavelength Bragg's law must be fulfilled for the readout angle. Important parameters for holography with BR films like the recording processes available, the spectral relation of absorption and refraction and the modification of the BR photocycle by physico-chemical methods will be discussed and the advantages of BR variants for some selected applications will be

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exemplified with real-time interferometry, optical filtering and holographic pattern recognition. 5.1 Recording and readout of information Holographic storage where recorded information should be stable over a period of more than 10 years is not possible with the currently available BR types. The BR films described, however, are suitable for transient optical recording which is very important for optical processing. Holography records analogue information and therefore any destructive readout procedure is to be avoided since it diminishes the contrast of the hologram. This is valid in particular for the so-called B-type recording of holograms. Information recorded with light absorbed by the B state, e.g. yellow, stimulates the transition B -> M. A readout beam, e.g. red, initiates the same photoreaction B -» M and lowers the contrast. A solution to the problem of destructive readout has been found by using two features of BR. First, the information is stored in a photoactive intermediate. An example is to record information by photochemical conversion of the M to the B state (M-type recording). Although the photoactivity of other intermediates of the BR photocycle could also be employed (Balashov & Litvin, 1981) the spectral separation of the B and M state by about 160 nm and the possibility to stabilize the M state at room temperature give particular advantage to M-type recording. Second, phase-sensitive readout can be performed at wavelengths in the near infrared (670—800 nm) where the absorption of BR films is very low and therefore no photochemical transitions disturb reading (see section 5.2). In Fig. 5 a a simplified scheme of the BR photocycle is shown where the two photochemical steps B -* J and M -> B are indicated which are important for holographic recording. Identical intensity patterns (hatched areas in Fig. $b) produced by interference of two light beams are seen for both yellow light in Btype recording and blue light in M-type recording. In the B-type process the BR film is in the B state at all positions initially and the incident intensity pattern leads to a high population of M in the bright regions (Fig. 56, shaded areas) and does not change the local BR population in the dark areas. Due to the nonlinear recording properties of BR the population distribution induced in the BR film may be not linearly correlated to the intensity distribution (Fig. 56, lowest row). In M-type recording the BR film must be optically pumped first, e.g. with yellow light, in order to obtain a high and uniformly distributed M population. Only then does the incident blue light intensity pattern lead to a population grating in the BR film. Compared to the B-type process the M/B population distribution is inverse. The real grating formed in the film is of a different shape due to the different nonlinearity of the two-photon response in M-type recording. A numeric simulation was given by Hampp et al. (1990 a) and the nonlinear transmission of BR was also analysed by Korchemskaya et al. (1990) and Werner et al. (1990). M-type recording in principle leads to the same holographic gratings as B-type recording, but shows the following advantages. It is a photocontrolled (AB and AM)

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~ -*

B AM

/

K a.

M (b)

Intensity grating yellow

Intensity grating blue (yellow background)

Initial population distribution

M

M

B

B

M

M

B

B

M

M

B

B B type M type Fig. 5. Non-destructive recording and readout process for transient holograms in BR films. Simplified scheme of the photochemical conversions of BR (a) and comparison of the B-type and M-type recording processes (6) used in holography. Hatched areas represent intensity distributions in the intensity grating, shaded and white areas in population distributions correspond to the M and B concentrations, respectively. In the M-type recording process the pumping wavelength serves simultaneously as a readout wavelength. Therefore reading 'creates' and does not 'destroy' the hologram.

and optically gated (AB) process in the sense that the yellow light acts at the same time as a pumping and as reading beam. Furthermore, the yellow light beam is constructive not destructive during the readout process of the hologram. The sensitivity of M-type holograms also depends strongly on parameters influencing the M population. For a pure B ^ M system where a photochemical reaction B->M with a reaction constant k1 = ax / yellow and a reaction M -»B with k2 = a2 / b l u e and a thermal decay of M -• B with ks — T^1 the local M population is described by M= where Bo is the concentration of BR.

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-20

20 -|

~r 550 600 650 Wavelength (nm)

Fig. 6. Relation of the light-induced changes of the absorption coefficient (A) and of the refractive index ( 0 ) in a BR film containing the BR variant BRD96N. The broken line represents the theoretical refractive index change calculated from the measured absorption changes on the basis of the Kramers-Kronig relation. (Modified from Zeisel & Hampp, 199')

Two assumptions go into this calculation. The first is that a BR molecule which relaxed to the B state immediately can be again photochemically converted B -* J (Dancshazy et al. 1986). The second is that the photochemical transition from M -> B (Chernavskii et al. 1989; Groma et al. 1984) does not involve a long-living intermediate. In a BR system with prolonged M lifetime, i.e. k3