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Bacteriorhodopsin—Novel biomolecule for nano devices
Analytica Chimica Acta 568 (2006) 47–56
Bacteriorhodopsin—Novel biomolecule for nano devices P.C. Pandey ∗ Department of Applied Chemistry, Institute of Technology, Banaras Hindu University, Varanasi 221005, India Received 29 August 2005; received in revised form 31 October 2005; accepted 8 November 2005 Available online 20 January 2006
Abstract The aim of this article is to provide insight on the use of a biological molecule—bacteriorhodopsin (bR) having all the basic properties necessary for the assembly of nanoscale electronic devices. Recent developments made during last decade supported by key references are reviewed in this contribution. Major emphasis on bR-based observations conducted in our laboratory has been elaborated. Important issues concerning structure, widely accepted photocycle of bR has been summarized. The possibility of nano-devices emanating from this biomolecule is briefly presented. © 2005 Elsevier B.V. All rights reserved. Keywords: Molecular sensors; Photochemistry; Bacteriorhodopsin; Photosensor; Molecular electronics material
1. Introduction The introduction of image resolving power in animals which is restricted to mainly three phyla of animal kingdom—Mollusks, Arthropods and Vertebrates are equipped with unique property of life named to be eyes. These three phyla have focused on a basically similar protein structure and an identical light absorbing chromophores-11-cis-retinal which is responsible for generating an optic nerve impulse following photo-excitation leading to complex series of reactions more specifically via hyper-polarization of plasma membrane of the rod cell in retina. The introduction of this chromophore in living organisms has triggered for attaining the sensation of infinite universe through photo-electrochemical processes. Activation of nerve impulse through hyper-polarization to the order of 1 mV is sufficient in a dark-adopted retina and is accomplished via a series of complex biochemical cycle involving transduction-α peripheral membrane protein of the G-protein family. A single molecule of photo activated rhodopsin catalyzes the activation of up to 1000 transducin molecule that represent the initial stage in the amplification process. Since the discovery of bR in early 1970s [1] the conceptual simplicity and experimental advantages of bacteriorhodopsin have made this light-driven proton pump a testing ground for hypotheses of transport mechanisms and
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new experimental technologies. Apart from such a tremendous potentiality of this biomolecule, human made devices involving the power of rhodopsin has been one of the greatest attraction during last decade and outcomes have shown the possibility of generating nano devices, e.g., (a) material for optical information recording, (b) molecular sensors, (c) electronic switches, (d) gates, (e) biological transistors, (f) artificial retina. The objective of this review is to summarize basic understanding of this biomolecule through key researches and the development made in our laboratory. The power of microbes in introducing biotechnology is unbelievable. Apart from abundance variety of microbes to be exploited in targeted direction, one of the microbe of Halobacterium family using rhodopsin analogue for converting the light energy into an electrochemical proton gradient, which in turn is used for ATP production by ATP synthase and the pigment is named as bacteriorhodopsin. The present review focuses our discussion on this molecule into following sections. 1.1. Purple membrane The membrane patches present on the surface of Halobacterium strain is called the purple membrane. The membrane is basically protein and lipid in the ratio of 75:25 has potential selectivity for proton transport. The protein forms a hexagonal two-dimensional crystal consisting of bacteriorhodopsin trimers. The unit cell contains three monomeric bR molecules, 28 lipid molecules and 8410 water molecules, giving a total
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number of 23,783 atoms. The membrane protein can be easily isolated and could be processed into mass production as is required for biotechnological applications [2–9]. 1.2. Structure of bacteriorhodopsin Bacteriorhodopsin is a stable trans-membrane protein of Halobacterium, prefers to live in salt marshes and can survive even in bizarre environmental conditions. Rhodopsin is a visual purple membrane with molecular weight of 40,000 Da and it is the photoreceptor protein present in the rods of retina of the eyes. It is composed of cis-retinol bound to protonated Schiff’s base to lysine moiety in the appropriate protein. This pigment has been prepared by Wald and Brown in 1950 [10]. Hubbard and Wald were awarded Noble Prize (1951) for this contribution. Isolated and completely sequenced the gene encoding human rhodopsin was conducted by Nathans and Hogness in 1984 [11]. Retinal photoreceptor protein of bipartite structure consists of the transmembrane protein opsin and a light-sensitive chromophore 11-cis-retinal, linked to opsin via a protonated Schiff base. Opsins are species specific proteins that form the basis of visual pigments integrated into rods and cones in the eye. There have been extensive investigations on identifying the structure of bacteriorhodopsin [12–21]. Model for the structure of bacteriorhodopsin based on highresolution electron cryomicroscopy was first studied by Henderson et al. in 1990 [22]. The retinal proteins from Halobacterium is configured into a seven-transmebrane helix (named as A, B, C, D, E, F and G) topology with short interconnecting loops. The helices are arranged into arc-like structure tightly surrounding a retinal molecule that is covalently bound via a Schiff base to a conserved lysine (Lys-216) on helix G. Retinal separates a cytoplasmic surface from an extra cellular half channel that is lined by amino acids crucial for efficient proton transport by bR (especially Asp-96 in the cytoplasmic and Asp-85 in the extracellular half channel). The Schiff base between retinal and Lys-216 is located at the center of this channel. For proceeding vectorial proton transport, de- and re-protonation of the Schiff base must occur from different sides of the membrane. Thus, the accessibility of the Schiff base for Asp-96 and Asp-85 must be switched during the catalytic cycle. The geometry of the retinal, the protonation state of the Schiff base, and its precise electrostatic interaction with the surrounding charges (Asp-85, Asp-212, Arg-82) and dipoles tune the absorption maximum to fit its biological function. 1.3. Photo cycle of bacteriorhodopsin It has been known from the earliest works that the photo cycle contains the spectrally distinguishable quasi-stable states K, L, M, N and O. Once K is produced from J, which arises from the excited state, the rest of the reaction cycle is a sequence of thermal reactions, as in other transport systems. Its kinetic description is therefore, of necessity, the point of departure for any proposed transport mechanism. The photocycle of bR has been extensively studied and being studied in order to understand the complex city of biological functions. There have been enor-
mous articles available on bacteriorhodopsin photo cycle and several of others are coming up in the literature each day. Some of the representative publications are listed in this review [23–29]. The kinetics of photocycle steps could be categorized into: (i) primary reaction, (ii) from the K590 to the L550 intermediate, (iii) first proton translocation step: from L550 to M410, (iv) first accessibility switch reaction from extracellular to cytoplasmic, (v) second proton transfer step: from M410 to N560, (vi) reisomerization of retinal from 13-cis to all-trans: N560 to O640, (vii) second accessibility switch reaction from cytoplasmic to extracellular: O640 to bR. In a stereoselective process, all-trans retinal is photoisomerized to 13-cis-retinal and discussed in following sections. 1.4. Nano devices based on bacteriorhodopsin 1.4.1. Molecular motor Molecular motors are a nano devices and can be made to work everywhere. The use of bacteriorhodopsin for making molecular motor is although hypothetical at this scenario however the photo induced proton gradient in coupling with cytochrome oxidase which moves proton using high energy electron could be utilized in generating electromotive force [30]. 1.4.2. Molecular transistors The possibility of introducing revolutionary change in the semiconductor industry is now visible within next few years. The introduction of system, called to be molecular electronics, will convert today’s computers into as little calculators. When the light velocity is significantly higher than the electron velocity, it is possible to create analogues of diodes and transistors based on photonic crystals with operation rates of up to 105 GHz [31]. The investigations made in the group of Dr. Weetall at NIST by Nikolai Vsevolodov et al. first produced an imaging device and microfilm made from biological materials called Biochrom film [32,33]. The key substance was bacteriorhodopsin and subsequently several attempts to produce imaging and information storage devices using biological materials have been published. Many of these publications have come from the laboratory of Robert Birge where he has developed a three-dimensional information storage device that incorporates bacteriorhodopsin as the storage element [34]. No doubt the investigation conducted so far involving biological molecules have generated beam of insight revealing future into molecular electronics age. 1.4.3. Molecular gates The photo induced variation in bacteriorhodopsin’s color with light: purple to yellow, then back to purple may make possible to serve as a switch, similar to an on–off light switch or the 0–1 logic gates in computers. The light energy of the bacteriorhodopsin can be converted to electrical energy, which is the key to its utility [35]. Investigations are being made in this direction and fruitful progress is awaited. 1.4.4. Artificial retina Laser light of two different wavelengths on the protein molecule can switch it back and forth between its purple and
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yellow forms. This behavior has prompted research on the use of bacteriorhodopsin as the element in artificial retinas and as memory or processing units in protein-based or optical computers. Artificial retinas based on the light transducing photoelectric protein bacteriorhodopsin exhibit differential responsivity, edge enhancement and motion detection. Under appropriate conditions, these artificial receptors mimic the differential responsivity characteristic of mammalian photoreceptor cells. The use of orientated bacteriorhodopsin to generate the photoelectrical signal provides rapid responsivity, high quantum efficiency and offers the potential of directly coupling the protein response to charge-sensitive semiconductor arrays. The ability to manipulate the properties of the protein via chemical and genetic methods enhances design flexibility [36,37]. The differential responsivity is the essential function in mobile target detection and edge pattern perception in human retina. A model array sensor (256 pixels) with SnO2 –aqueous gel junction showed that visual information processing’s for mobile image extraction are performed within 100 s, far faster than computer-assisted image processing, at the material level without the need of differential circuitry and external dc driving power for the sensor. The use of chitin gel as an electrolyte proved to protect bR from biological decay, allowing the sensor to function for years. For ultimate end of the application, however, molecular wiring to the molecule of bR to elicit its charge displacement and proton transfer events is the key subject, common to the implementation of molecular electronic devices that surpass the silicon-based ones. One for this end may be implantation of the photoelectric bR layer on the retinal membrane for the purpose of visual prosthesis for those who lack the sensory retinal but retain the neural network [38,39]. 1.4.5. Molecular sensors The absorption of a light quantum by the retinal leads to reversible color changes from purple to yellow due to protein conformational changes, the molecule finally returning to the ground state and the process follows displacements of the optical absorption bands. For practical sensing applications, one can monitor the spectral and kinetic parameters of films made from bR incorporated into a matrix. Consideration of the question of external influences (e.g., due to the presence of ambient chemical substances) on the bR photo chromic processes is of prime importance. By changing external conditions (temperature, hydrostatic pressure, electrical field intensity, etc.), one can influence the photo induced processes of the light-sensitive retinal–protein complex. The chemical environment of bR also influences the photo cycle parameters [40]. For example, humidity [41] and [42], solutions of local anesthetics [43], organic vapors [44], and ammonium ions and amines [45,46] and all have demonstrable effects on the spectral and kinetic parameters. The effect of pH has also been studied [47]. Inclusion of halogen-, nitrogen- and sulphur-containing substances (called chemical additives) in various combinations into the film is a way to permanently modify the photosensitive and temporal characteristics of the photo cycle over a wide range [41,48,49,50], with the possibility of enhancing the chemical sensitivity and selectivity of the film. Recent report on bR-based chemical sens-
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ing was demonstrated by Sharkany et al. [41] for humidity and ammonia. When increasing the ambient relative humidity from 12 to 85%, the optical absorption of the films incorporating a chemical additive doubled, and the temporal characteristics of the photo cycle varied by almost three orders of magnitude. On the other hand, the presence of ammonia influenced the photo cycle parameters to a similar extent in an additive-free film. In both cases, the changes were completely reversible. It is thereby demonstrated that bR is a good candidate for a universal material for making the sensitive elements of fiber and integrated optical sensors and that the chemical additives can be used to confer selectivity on the response. Weetall et al. have made extensive investigations on bR-based technology with special attention to dehydration of bR modified film [51], stabilization of bR in sol–gel glass [46,52], optical and electrical characterization of bacteriorhodopsin films [53], measurement of proton release and uptake by analogues of bacteriorhodopsin [54] and photo transformation and proton pumping activity of the 14-fluorobacteriorhodopsin derivatives [55]. His group has been remained active on developing bR-based nano devices using bR and its analogue. By nature, many biological macromolecules are highly efficient at recognizing specific analytes or catalyzing reactions in aqueous biological media. It has been shown that silicate glasses obtained by the sol–gel method can provide such a host matrix and that biomolecules immobilized by this method retain their functional characteristics to a large extent. These bio-compatible glasses make it possible to retain the specificity and reactivity of biological molecules in the solid-state and provide morphological and structural control that is not available when the biological molecules are simply dissolved in aqueous media. Furthermore, the amorphous nature of the glassy material does not impart a geometric order to the entrapped biomolecules, many of the characteristics of the liquid state are retained despite the fact that the molecule is trapped in a solid material. We have recently reported the use of organically modified sol–gel glass (ormosil) for encapsulation/sandwiching of targeted molecules [56] and found relatively better stability followed by ormosil’s immobilization of the same. These ormosils differ from silicate glasses with respect to the presence of desired reactive organic functional group in sol–gel precursors which are even intact even after the formation of nano-structured solid-state network. The presence of such organic functionality could be useful in emanating valuable information on specific interaction of encapsulated biomolecule as well as for anchoring additional/same targeted moieties within nano-structured geometry. Accordingly, we intended to stabilize bR using ormosil and examine the photo-electrochemistry under this condition. Three alkoxysilane precursors having organic functionalities namely 3-aminopropyltrimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane and phenyltrimethoxysilane are chosen in the present investigation. The presence of amino-group as well as epoxy-group within ormosil network might undergo spontaneous interaction with targeted biomolecule that may cause, same or both promoted or inhibited biological activity after encapsulation/stabilization of the biological molecule. Some important findings on D96N mutant
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bR-systems have been studied in our laboratory and important findings are given in this communication. 2. Experimental D96N mutant bacteriorhodopsin was used in this investigation. Phenyltrimethoxysilane, 3-aminopropyltrimethoxysilane and 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane were obtained from Aldrich Chemical Co. All other chemicals used were of analytical grade. Tris–HCl buffer (10 mM, pH 7.0) was used as working medium. 2.1. Measurement of UV–vis spectra The bR preparation was studied by measuring the absorbance of bR on quartz plate/or on ATO electrode or in solution between 370 and 700 nm in absence and the presence of external light source using a double beam UV–vis spectrophotometer (Systronics, India, mode 2201). 2.2. Electrochemical measurement of immobilized bR on ATO electrode The electrochemical measurements were made using immobilized bR on ATO electrode as described earlier [57]. The electrochemical measurements were performed with a Solartron Electrochemical Interface (model 1287, Solartron, U.K.) connected to a PC through the serial port. The electrode response
was recorded and plotted with a Pinter. A one compartment home made cell with a working volume of 5 ml and a bR immobilized ATO electrode, platinum foil auxiliary electrode and Ag/AgCl reference electrodes were used for the measurements. The electrochemical cell as described earlier was used as such [58]. However, the light source (Fig. 1a and b) was re-designed to get relatively better stability. A device to expose the working electrode with external light source (Fig. 1a and b) was made which was attached to the back portion of transparent Perspex sheet onto which a working electrode was placed into the electrochemical cell with a three electrode configuration (Fig. 1c), with the arrangement of blue (Fig. 1a) and yellow (Fig. 1b) filters between working electrode and light source. The light source was a 50 W small light bulb placed within reflecting mirror designed in such a way that the transmitted light of an angle of 25◦ . The electrochemical measurements were made by polarizing the working electrode at desired potentials followed by the measurement of steady-state background current. Subsequently, the working electrode was exposed to light using a blue filter followed by a yellow filter. The photo-current as a function of time was recorded and plotted. The photo-current was measured at different potentials and with the light expose to the working electrode for sufficient duration to reach steady value followed by switching off the light exposure. At the optimum potential, the photo-current as a function of time was measured in the presence of varying concentrations of arginine and ammonium chloride. The experiments were performed in Tris–HCl buffer (10 mM, pH 7.0) and at 25 ◦ C.
Fig. 1. Home made photoelectrochemical cell and light source equipped with yellow and blue filters. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 3. (A) Spectra of bacteriorhodopsin in solution: curve 1 represents the absorption spectra in dark whereas curves 2, 3 and 4 represent the same in the presence of external yellow light [2 = control, 3 = in the presence of ammonium ion and 4 = in the presence of benzylamine]. (B) Absorption spectra of bacteriorhodopsin in solid-state. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) Fig. 2. Widely accepted photocycle of bacteriorhodopsin.
3. Results and discussion 3.1. Photosensitivity of bacteriorhodopsin The power of rhodopsin in image resolving eye is amazing and the technology behind that is perfect till the existing limitation of brain simulation. The knowledge available so far reveals that the generation of this perfect technology is basically due to integrated function of chromophore having close to 100% quantum efficiency together most efficient signal transduction in co-ordinated neural network justifying the existence of nanoelectronics in visual reception. Nevertheless, we have been trying to explore the possibility of generating molecular sensors and artificial retina based on optoelectrochemistry of bR within our resources. Fortunately, we have been able to record the sensitivity of bR photocycle on the presence of amine-residue and ammonium ion first time. The recordings of our observations were not concerned to fast kinetics of the bR photo cycle and basically concerns the observation recorded in finite time in dark and light. Before going ahead on this finding, it is now important to have a quick look of the photo cycle consisting of fast and slow kinetic part of bR as shown in Fig. 2. The cycle can be illustrated into six steps having major components of: (1) isomerization, (2) ion transport and (3) accessibility change. On photo-excitation of the chromophore, it first isomerizes from an all-trans to a 13cis configuration (step 1) followed by a proton transfer from the Schiff base to the proton acceptor Asp-85 (step-2). The proton transfer ultimately referred as to be de-protonation step (iontransfer step part-2) leads the formation of M-state. The Schiff base existed on M-state is then reprotonated from Asp-96 from the cytoplasmic surface and retinal reisomerizes thermally and the accessibility of the Schiff base switches back to extra cellular to reestablish the initial state (accessibility change, step-3). The
accessibility is switched from extra cellular to intracellular and thus the presentation of M-state requires inversion in intracellular to extra cellular direction. Fig. 2 justify the most accepted photo cycle of bR based on the data on fast and slow kinetics of the photocycle. We have tried to understand the slow part of the photo cycle as the existence of bR in two state might leads to the development of many molecular devices to be referred as practical molecular electronics materials as mentioned in the preceding sections. Accordingly, we proceeded to record the spectra of bR in light and dark to examine the stability of the same for practical applications. The results are recorded in Fig. 3A in dark (1), in the presence of external light (2), in the presence of external light and ammonium chloride (3) and in the presence of external light and benzylamine (4). The spectra were recorded in 10 mM phosphate buffer pH 7.0. The spectra clearly demonstrate the dependence of M-state on the presence of these chemical and opens the possibility for the exploitation of bR in the development of molecular sensors. In this direction, we further attempted to study the photochemistry of bR in solid-state on ATO electrode for its exploitation in opto-electrochemistry. Fig. 3B shows the spectra of bR in solid-state on ATO electrode and justify that suitability of the material in solid-state on ATO surface (Fig. 3B). The above preliminary finding directed our attention to investigate the photo activity of bR under following conditions: (i) on-line probing of bR at 410 nm under dark followed by illuminating the bR in external light and subsequently to record the decay kinetics at 410 nm after shutting down the external light source, (ii) on-line probing of bR at 530 nm under dark followed by illuminating the bR in external light and subsequently to record the decay kinetics at 530 nm after shutting down the external light source, (iii) on-line probing of bR at 640 nm under dark followed by illuminating the bR in external light and subsequently to record the decay kinetics at 640 nm after shutting
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Fig. 4. The kinetics of decay of the D96N mutant at 410, 530 and 640 nm (1) control, (2) in the presence of ammonium ion (0.003 M) and (3) in the presence of benzylamine (0.04 M) in 0.01 M phosphate buffer pH 7.0. The initial data recorded between 0 and 20 s (region i) represent the absorption in absence of external light data recorded between 20 and 40 s (region if) show the absorbance in the presence of external light whereas the rest of the curve (40–60 s; region iii) shows the absorption change in dark, back to the ground state (bR, 0.25 mg/ml).
down the external light source [47]. Such recordings are shown in Fig. 4. Interesting observations on slow kinetics of bR have been recorded and trigger the use of bR in molecular sensing based on kinetic data. These recordings have been evaluated in following three type of analysis namely (a) half time decay parameter at 410, 530 and 640 nm under control, in the presence of ammonium ion and in the presence of benzylamine, (b) total absorption at 410, 530 and 640 nm under control, in the presence of ammonium ion and in the presence of benzylamine and (c) absorption recorded in 500 ms at 410, 530 and 640 nm under control, in the presence of ammonium ion and in the presence of benzylamine. These findings are shown in Fig. 5. The
results again justify remarkable dependence of slow kinetics of bR photocycle on such chemicals. 3.2. Probing of enzymatic reaction The photocycle of bR has been susceptible to the presence of proton concentration even in wild type however become more apparent in D96N mutant bR. D96N mutant bacteriorhodopsin has been used in this communication which differs from the wild type by exchanging 96-residue of amino acid using a non-protonated residue. Such exchange perturbs the auto protonexchange ability of biomolecule that ultimately influences the
Fig. 5. Variation of the half time (T1/2 ), absorption of decay kinetics of decay kinetics and variation of the absorption of decay kinetics recorded at 410, 530 and 640 nm within the initial 500 ms recorded at 410, 530 and 640 nm of D96N mutant bR (0.25 mg/ml): (1) control, (2) in the presence of ammonium ion (0.003 M) and (3) in the presence of benzylamine (0.04 M) in 0.01 M phosphate buffer pH 7.0.
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Fig. 6. (A) The effect of enzymatic reaction kinetic of M-state decay of D96N bacteriorhodopsin at 410. The initial data recorded in 0–5 s represent the absorption in light whereas the rest of the decay curve shows absorption in dark back to ground state: 1 = control, 2 = in the presence of urease (0.3 mg/ml) and urea 0.02 M, 3 = in the presence of acetyl cholinesterase (0.08 mg/ml) and acetylcholine 0.02 M. (B) Variation in half time decay (T1/2 ) of D96N bacteriorhodopsin in the presence of increasing concentrations of benzylamine and ammonium ion (bR = 0.31 mg/ml).
kinetics of de-protonation and re-protonation processes of the photo cycle. Accordingly, the photochemistry of bR becomes dependent on the proton concentration present extra cellular side of bio-membrane. We have been able to record the probing of enzymatic reactions first time based on monitoring the slower kinetics of photo cycle [46]. The results as shown in Fig. 6 show the slower kinetic part (M-state decay) as a function of acetyl cholinesterase (AChE)- and urease-catalyzed reactions. The absorbance at 412 nm was recorded in the presence of varying concentrations of respective substrates [control (Fig. 6A, curve 1), urea (Fig. 6A, curve 2) and acetylcholine (Fig. 6A, curve 3). The measurement of decay kinetics at 412 nm was made. Again the results justify the dependence of photo cycle on the alternation proton concentration generated out side the extra cellular fluid. Urease catalyzed reaction generates base and thus causes reduction in proton outside extra cellular side whereas acetyl cholinesterase (AChE) catalyzed reaction generates acid thus increases the proton concentration under similar concentration. However, urease catalyzed reaction also produces NH4 + ion that really influences the photo cycle. Accordingly, the direct effect of pH variation could not be evaluated. The data in Fig. 6B shows inversion in kinetics of slow part of the photo cycle in the presence of benzylamine and ammonium chloride. The measurements were again conducted at 412 nm similar to that of recorded for Fig. 6A. 3.3. Photo-electrochemical investigations on bR photo cycle Since the bacteriorhodopsin is a highly sensitive photoactive material, the optical transparency of the immobilized matrix is one of the important requirements for practical applications. Apart from many matrices used so far, the matrix comparable to optical glass could be more suited as one of the option for practical applications. Accordingly, sol–gel glasses, com-
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parable to conventional optical material, made under specific conditions and derived from alkoxysilane precursors through sol–gel process is getting introduced not only for specific applications in studying bR photochemistry but to cover wide range of optical materials as well. Introduction of organic functionality in alkoxysilanes precursors, leading to organically modified sol–gel glass (ormosil) that might have additional advantage for anchoring targeted molecules and consequently making the process feasible to the production of functionalized optical materials for exploitation in many directions that has not been possible using conventional glasses so far. The ormosil derived from the functionalized alkoxysilane precursors at least trigger the introduction of such optically transparent material in studying the photochemistry of bacteriorhodopsin. Before providing details into the use of ormosil films in stabilizing the bR for opto-electrochemical investigations, we first attempted to study the photoelectrochemistry of bR present on ATO electrode and placed into electrochemical cell as shown in Fig. 1 or even as described earlier [58]. The results on photoelectrochemical responses are shown in Fig. 7. In this case, bR was simply adsorbed on ATO electrode and the membrane protein was not oriented. There is remarkable dependence of bipolar response of bR in dark and in the presence of light with significant dependence of photocurrent on applied potential. The home made light source as shown in Fig. 1 was used for exposure of bR on ATO electrode. The yellow light was allowed to pass though the working electrode for desired time. The recording as shown in Fig. 7A represents the data on photoelectrochemical response of un-oriented bR at −100 mV versus Ag/AgCl, Fig. 7B shows the same at 100 mV versus Ag/AgCl whereas Fig. 7C shows at −30 mV versus Ag/AgCl. On increasing the negative potential there is an increase in photocurrent whereas increase in positive potential reduces the magnitude of photocurrent. The recording shown in Fig. 7D and E justify the variation in bipolar behavior of photocurrent on altering the exposure of external light on bR. The external yellow light (Fig. 1) was exposed to bR sample for 10 s in case of Fig. 7D whereas the same was reduced to 1 s for Fig. 7E. There is transition from bipolar behavior when exposure time is reduced from 10 to 1 s. This preliminary observations although demonstrated in photo-sensing of targeted analyte (Fig. 8) however the finding provides an insight to use this molecule in designing artificial retina after suitably configuring the bR on optical material. After each set of photocurrent measurement, the bR was exposed to blue light. The data recorded in Fig. 8 shows the results on photocurrent at varying concentrations of the chemicals affecting the photo cycle. The electrode was polarized at −30 mV versus Ag/AgCl followed by exposure of the working electrode to external yellow light for 10 s. There is an increasing trend in both forward and backward photocurrent on increasing the lysine (Fig. 8a)/Arginine (Fig. 8b) concentration whereas the same shows decreasing trends when ammonium ions are present under similar conditions. It should be noted that in control (Fig. 8a) the response is bipolar however slightly less exposure time (manual error) might have caused diffused distinction of steady-state in control. Fig. 9 shows the typical recordings in the presence and absence of amine and ammonium ion by exposing the working
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Fig. 7. A typical recording of photocurrent at various applied potentials: (A) −100 mV, (B) +100 mV and (C) −30 mV vs. Ag/AgCl. The working electrode was exposed to yellow light for >12 s followed by recording in the dark as a function of time, (D) typical recording of the variation of photocurrent followed by exposure of working electrode for >12 s and subsequent recording in the dark as a function of time at −30 mV vs. Ag/AgCl. (E) A typical recording of the variation of photocurrent after exposure to light of working electrode for 1 s followed by subsequent recording in the dark as a function of time at −30 mV vs. Ag/AgCl. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
electrode for 1 s only. Even 1 s exposure time drastically altered the peak current in the presence of such analyte reflecting the possibility of exploitation of the bR in molecular photo-sensor designing. It appears that proton translocation sites linked to faster portion of photo cycle are kinetically influenced in the
Fig. 8. (a) A typical recording of the variation of photocurrent followed by exposure of working electrode for >12 s and subsequent recording in the dark as a function of time at −50 mV vs. Ag/AgCl in the absence (control) and presence of lysine (0.01, 0.02 and 0.03 M). (b) A typical recording of the variation of photocurrent followed by exposure of working electrode for >12 s and subsequent recording in the dark as a function of time at −50 mV vs. Ag/AgCl in the presence of arginine 0.05 M. The two insets show the result on control and in the presence of arginine (0.01 M).
presence of these amino- and ammonium residues, however, the mechanism of such interaction require thorough investigation and originate from the exceptional properties of proton dynamics which caused the existence of life on earth. These residues possibly affect the charge density near the retinal induced by its photo-isomerization followed by extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pKa values. The next stage of the investigation was concerns to coupling the bR within ormosil films and concerns to retain the photo activity of bR within isolated transparent matrixes. As a trial, we have worked on sandwiching bR using ormosil derived from phenyltrimethoxysilane, 3-aminopropyltrimethoxysilane and 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane. The preliminary results demonstrated interesting observations for practical usability of the material in technological development. The detailed output would be presented in a later publication.
Fig. 9. Typical recordings of photocurrent for the exposure of working electrode for 1 s at −30 mV vs. Ag/AgCl under various conditions: (a) control recording in the absence of amine and ammonium chloride, (b) in the presence of ammonium chloride and (c) in the presence of cysteine (0.015 M).
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4. Conclusion The present article demonstrates the science of bacteriorhodopsin which has potentiality in generating nano-devices with key references supported by the contributions made in our laboratory. The structure, habitat, properties of this biomolecule are discussed. The examples of nano-devices possibly emanating form bR have also been discussed. Attentions have been made on the development of bR-based nano-devices, i.e., molecular transistor, molecular motor, molecular gate, artificial retina and molecular sensors. Some important finding based on spectroscopy and photo-electrochemistry conducted in our laboratory has been summarized. The sensitivity of M-state to the presence of some amine and amino-compounds are reported based on spectroscopic and photo electrochemical observations. Both observations reveal that the presence of amine causes increase in life M-state whereas ammonium-residue affect in opposite fashion. The photoelectrochemical response of bR is found sensitive to the magnitude and polarity of applied voltage. In the presence of light, the biomolecule shows forward photocurrent whereas in absence of the same backward photocurrent is recorded. An increase in negative polarization potential result increase in forward and backward photocurrent. The bipolar response recorded on electrochemical polarization of bR molecule in the presence and absence of light and amine-/ammonium-residues has been reported reflecting its novelty in possible designing of artificial retina. The duration of light exposure time on photo-electrochemical bipolar response of bR are also reported that provide valuable information for technological exploitation of bR molecule in several other directions. Acknowledgement The author is thankful to DST, New Delhi for financial support. References [1] D. Osterhelt, W. Stoeckenius, Proc. Natl. Acad. Sci. U.S.A. 70 (1973) 2853. [2] D. Oesterhelt, W. Stoeckenius, Methods Enzymol. 31 (Pt. A) (1974) 667–678. [3] R. Henderson, P.N. Unwin, Nature 257 (1975) 28–32. [4] L.O. Essen, R. Siegert, W.D. Lehmann, D. Oesterhelt, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 11673–11678. [5] H. Belrhali, P. Nollert, A. Royant, C. Manzel, J.P. Rosenbusch, E.M. Landau, E. Pebay-Peyroula, Struct. Fold. Des. 7 (1999) 909–917. [6] M.B. Jackson, J.M. Sturtevant, Biochemistry 17 (1978) 911–991. [7] V.I. Shnvrov, P.L. Mateo, FEBS Lett. 324 (1993) 237–240. [8] K. Hiraki, T. Hamanaka, T. Mitsui, Y. Kito, Biochim. Biophys. Acta 647 (1981) 18–28. [9] M. Rehorek, M.P. Heyn, Biochemistry 18 (1979) 4977–4983. [10] G. Wald, P.K. Brown, Proc. Natl. Acad. Sci. U.S.A. 36 (1950) 84. [11] J. Nathans, D.S. Hogness, Proc. Natl. Acad. Sci. U.S.A. 81 (1984) 4851. [12] R.A. Bogomolni, R.A. Baker, R.H. Lozier, W. Stoeckenius, Biochim. Biophys. Acta 440 (1976) 68–88. [13] L. Lasse, F. Michael, P. Jussi, P. Sinikka, T. Jaaskelainen, Opt. Mater. 27 (2004) 57–62.
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Bacteriorhodopsin—Novel biomolecule for nano devices P.C. Pandey ∗ Department of Applied Chemistry, Institute of Technology, Banaras Hindu University, Varanasi 221005, India Received 29 August 2005; received in revised form 31 October 2005; accepted 8 November 2005 Available online 20 January 2006
Abstract The aim of this article is to provide insight on the use of a biological molecule—bacteriorhodopsin (bR) having all the basic properties necessary for the assembly of nanoscale electronic devices. Recent developments made during last decade supported by key references are reviewed in this contribution. Major emphasis on bR-based observations conducted in our laboratory has been elaborated. Important issues concerning structure, widely accepted photocycle of bR has been summarized. The possibility of nano-devices emanating from this biomolecule is briefly presented. © 2005 Elsevier B.V. All rights reserved. Keywords: Molecular sensors; Photochemistry; Bacteriorhodopsin; Photosensor; Molecular electronics material
1. Introduction The introduction of image resolving power in animals which is restricted to mainly three phyla of animal kingdom—Mollusks, Arthropods and Vertebrates are equipped with unique property of life named to be eyes. These three phyla have focused on a basically similar protein structure and an identical light absorbing chromophores-11-cis-retinal which is responsible for generating an optic nerve impulse following photo-excitation leading to complex series of reactions more specifically via hyper-polarization of plasma membrane of the rod cell in retina. The introduction of this chromophore in living organisms has triggered for attaining the sensation of infinite universe through photo-electrochemical processes. Activation of nerve impulse through hyper-polarization to the order of 1 mV is sufficient in a dark-adopted retina and is accomplished via a series of complex biochemical cycle involving transduction-α peripheral membrane protein of the G-protein family. A single molecule of photo activated rhodopsin catalyzes the activation of up to 1000 transducin molecule that represent the initial stage in the amplification process. Since the discovery of bR in early 1970s [1] the conceptual simplicity and experimental advantages of bacteriorhodopsin have made this light-driven proton pump a testing ground for hypotheses of transport mechanisms and
∗
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new experimental technologies. Apart from such a tremendous potentiality of this biomolecule, human made devices involving the power of rhodopsin has been one of the greatest attraction during last decade and outcomes have shown the possibility of generating nano devices, e.g., (a) material for optical information recording, (b) molecular sensors, (c) electronic switches, (d) gates, (e) biological transistors, (f) artificial retina. The objective of this review is to summarize basic understanding of this biomolecule through key researches and the development made in our laboratory. The power of microbes in introducing biotechnology is unbelievable. Apart from abundance variety of microbes to be exploited in targeted direction, one of the microbe of Halobacterium family using rhodopsin analogue for converting the light energy into an electrochemical proton gradient, which in turn is used for ATP production by ATP synthase and the pigment is named as bacteriorhodopsin. The present review focuses our discussion on this molecule into following sections. 1.1. Purple membrane The membrane patches present on the surface of Halobacterium strain is called the purple membrane. The membrane is basically protein and lipid in the ratio of 75:25 has potential selectivity for proton transport. The protein forms a hexagonal two-dimensional crystal consisting of bacteriorhodopsin trimers. The unit cell contains three monomeric bR molecules, 28 lipid molecules and 8410 water molecules, giving a total
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number of 23,783 atoms. The membrane protein can be easily isolated and could be processed into mass production as is required for biotechnological applications [2–9]. 1.2. Structure of bacteriorhodopsin Bacteriorhodopsin is a stable trans-membrane protein of Halobacterium, prefers to live in salt marshes and can survive even in bizarre environmental conditions. Rhodopsin is a visual purple membrane with molecular weight of 40,000 Da and it is the photoreceptor protein present in the rods of retina of the eyes. It is composed of cis-retinol bound to protonated Schiff’s base to lysine moiety in the appropriate protein. This pigment has been prepared by Wald and Brown in 1950 [10]. Hubbard and Wald were awarded Noble Prize (1951) for this contribution. Isolated and completely sequenced the gene encoding human rhodopsin was conducted by Nathans and Hogness in 1984 [11]. Retinal photoreceptor protein of bipartite structure consists of the transmembrane protein opsin and a light-sensitive chromophore 11-cis-retinal, linked to opsin via a protonated Schiff base. Opsins are species specific proteins that form the basis of visual pigments integrated into rods and cones in the eye. There have been extensive investigations on identifying the structure of bacteriorhodopsin [12–21]. Model for the structure of bacteriorhodopsin based on highresolution electron cryomicroscopy was first studied by Henderson et al. in 1990 [22]. The retinal proteins from Halobacterium is configured into a seven-transmebrane helix (named as A, B, C, D, E, F and G) topology with short interconnecting loops. The helices are arranged into arc-like structure tightly surrounding a retinal molecule that is covalently bound via a Schiff base to a conserved lysine (Lys-216) on helix G. Retinal separates a cytoplasmic surface from an extra cellular half channel that is lined by amino acids crucial for efficient proton transport by bR (especially Asp-96 in the cytoplasmic and Asp-85 in the extracellular half channel). The Schiff base between retinal and Lys-216 is located at the center of this channel. For proceeding vectorial proton transport, de- and re-protonation of the Schiff base must occur from different sides of the membrane. Thus, the accessibility of the Schiff base for Asp-96 and Asp-85 must be switched during the catalytic cycle. The geometry of the retinal, the protonation state of the Schiff base, and its precise electrostatic interaction with the surrounding charges (Asp-85, Asp-212, Arg-82) and dipoles tune the absorption maximum to fit its biological function. 1.3. Photo cycle of bacteriorhodopsin It has been known from the earliest works that the photo cycle contains the spectrally distinguishable quasi-stable states K, L, M, N and O. Once K is produced from J, which arises from the excited state, the rest of the reaction cycle is a sequence of thermal reactions, as in other transport systems. Its kinetic description is therefore, of necessity, the point of departure for any proposed transport mechanism. The photocycle of bR has been extensively studied and being studied in order to understand the complex city of biological functions. There have been enor-
mous articles available on bacteriorhodopsin photo cycle and several of others are coming up in the literature each day. Some of the representative publications are listed in this review [23–29]. The kinetics of photocycle steps could be categorized into: (i) primary reaction, (ii) from the K590 to the L550 intermediate, (iii) first proton translocation step: from L550 to M410, (iv) first accessibility switch reaction from extracellular to cytoplasmic, (v) second proton transfer step: from M410 to N560, (vi) reisomerization of retinal from 13-cis to all-trans: N560 to O640, (vii) second accessibility switch reaction from cytoplasmic to extracellular: O640 to bR. In a stereoselective process, all-trans retinal is photoisomerized to 13-cis-retinal and discussed in following sections. 1.4. Nano devices based on bacteriorhodopsin 1.4.1. Molecular motor Molecular motors are a nano devices and can be made to work everywhere. The use of bacteriorhodopsin for making molecular motor is although hypothetical at this scenario however the photo induced proton gradient in coupling with cytochrome oxidase which moves proton using high energy electron could be utilized in generating electromotive force [30]. 1.4.2. Molecular transistors The possibility of introducing revolutionary change in the semiconductor industry is now visible within next few years. The introduction of system, called to be molecular electronics, will convert today’s computers into as little calculators. When the light velocity is significantly higher than the electron velocity, it is possible to create analogues of diodes and transistors based on photonic crystals with operation rates of up to 105 GHz [31]. The investigations made in the group of Dr. Weetall at NIST by Nikolai Vsevolodov et al. first produced an imaging device and microfilm made from biological materials called Biochrom film [32,33]. The key substance was bacteriorhodopsin and subsequently several attempts to produce imaging and information storage devices using biological materials have been published. Many of these publications have come from the laboratory of Robert Birge where he has developed a three-dimensional information storage device that incorporates bacteriorhodopsin as the storage element [34]. No doubt the investigation conducted so far involving biological molecules have generated beam of insight revealing future into molecular electronics age. 1.4.3. Molecular gates The photo induced variation in bacteriorhodopsin’s color with light: purple to yellow, then back to purple may make possible to serve as a switch, similar to an on–off light switch or the 0–1 logic gates in computers. The light energy of the bacteriorhodopsin can be converted to electrical energy, which is the key to its utility [35]. Investigations are being made in this direction and fruitful progress is awaited. 1.4.4. Artificial retina Laser light of two different wavelengths on the protein molecule can switch it back and forth between its purple and
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yellow forms. This behavior has prompted research on the use of bacteriorhodopsin as the element in artificial retinas and as memory or processing units in protein-based or optical computers. Artificial retinas based on the light transducing photoelectric protein bacteriorhodopsin exhibit differential responsivity, edge enhancement and motion detection. Under appropriate conditions, these artificial receptors mimic the differential responsivity characteristic of mammalian photoreceptor cells. The use of orientated bacteriorhodopsin to generate the photoelectrical signal provides rapid responsivity, high quantum efficiency and offers the potential of directly coupling the protein response to charge-sensitive semiconductor arrays. The ability to manipulate the properties of the protein via chemical and genetic methods enhances design flexibility [36,37]. The differential responsivity is the essential function in mobile target detection and edge pattern perception in human retina. A model array sensor (256 pixels) with SnO2 –aqueous gel junction showed that visual information processing’s for mobile image extraction are performed within 100 s, far faster than computer-assisted image processing, at the material level without the need of differential circuitry and external dc driving power for the sensor. The use of chitin gel as an electrolyte proved to protect bR from biological decay, allowing the sensor to function for years. For ultimate end of the application, however, molecular wiring to the molecule of bR to elicit its charge displacement and proton transfer events is the key subject, common to the implementation of molecular electronic devices that surpass the silicon-based ones. One for this end may be implantation of the photoelectric bR layer on the retinal membrane for the purpose of visual prosthesis for those who lack the sensory retinal but retain the neural network [38,39]. 1.4.5. Molecular sensors The absorption of a light quantum by the retinal leads to reversible color changes from purple to yellow due to protein conformational changes, the molecule finally returning to the ground state and the process follows displacements of the optical absorption bands. For practical sensing applications, one can monitor the spectral and kinetic parameters of films made from bR incorporated into a matrix. Consideration of the question of external influences (e.g., due to the presence of ambient chemical substances) on the bR photo chromic processes is of prime importance. By changing external conditions (temperature, hydrostatic pressure, electrical field intensity, etc.), one can influence the photo induced processes of the light-sensitive retinal–protein complex. The chemical environment of bR also influences the photo cycle parameters [40]. For example, humidity [41] and [42], solutions of local anesthetics [43], organic vapors [44], and ammonium ions and amines [45,46] and all have demonstrable effects on the spectral and kinetic parameters. The effect of pH has also been studied [47]. Inclusion of halogen-, nitrogen- and sulphur-containing substances (called chemical additives) in various combinations into the film is a way to permanently modify the photosensitive and temporal characteristics of the photo cycle over a wide range [41,48,49,50], with the possibility of enhancing the chemical sensitivity and selectivity of the film. Recent report on bR-based chemical sens-
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ing was demonstrated by Sharkany et al. [41] for humidity and ammonia. When increasing the ambient relative humidity from 12 to 85%, the optical absorption of the films incorporating a chemical additive doubled, and the temporal characteristics of the photo cycle varied by almost three orders of magnitude. On the other hand, the presence of ammonia influenced the photo cycle parameters to a similar extent in an additive-free film. In both cases, the changes were completely reversible. It is thereby demonstrated that bR is a good candidate for a universal material for making the sensitive elements of fiber and integrated optical sensors and that the chemical additives can be used to confer selectivity on the response. Weetall et al. have made extensive investigations on bR-based technology with special attention to dehydration of bR modified film [51], stabilization of bR in sol–gel glass [46,52], optical and electrical characterization of bacteriorhodopsin films [53], measurement of proton release and uptake by analogues of bacteriorhodopsin [54] and photo transformation and proton pumping activity of the 14-fluorobacteriorhodopsin derivatives [55]. His group has been remained active on developing bR-based nano devices using bR and its analogue. By nature, many biological macromolecules are highly efficient at recognizing specific analytes or catalyzing reactions in aqueous biological media. It has been shown that silicate glasses obtained by the sol–gel method can provide such a host matrix and that biomolecules immobilized by this method retain their functional characteristics to a large extent. These bio-compatible glasses make it possible to retain the specificity and reactivity of biological molecules in the solid-state and provide morphological and structural control that is not available when the biological molecules are simply dissolved in aqueous media. Furthermore, the amorphous nature of the glassy material does not impart a geometric order to the entrapped biomolecules, many of the characteristics of the liquid state are retained despite the fact that the molecule is trapped in a solid material. We have recently reported the use of organically modified sol–gel glass (ormosil) for encapsulation/sandwiching of targeted molecules [56] and found relatively better stability followed by ormosil’s immobilization of the same. These ormosils differ from silicate glasses with respect to the presence of desired reactive organic functional group in sol–gel precursors which are even intact even after the formation of nano-structured solid-state network. The presence of such organic functionality could be useful in emanating valuable information on specific interaction of encapsulated biomolecule as well as for anchoring additional/same targeted moieties within nano-structured geometry. Accordingly, we intended to stabilize bR using ormosil and examine the photo-electrochemistry under this condition. Three alkoxysilane precursors having organic functionalities namely 3-aminopropyltrimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane and phenyltrimethoxysilane are chosen in the present investigation. The presence of amino-group as well as epoxy-group within ormosil network might undergo spontaneous interaction with targeted biomolecule that may cause, same or both promoted or inhibited biological activity after encapsulation/stabilization of the biological molecule. Some important findings on D96N mutant
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bR-systems have been studied in our laboratory and important findings are given in this communication. 2. Experimental D96N mutant bacteriorhodopsin was used in this investigation. Phenyltrimethoxysilane, 3-aminopropyltrimethoxysilane and 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane were obtained from Aldrich Chemical Co. All other chemicals used were of analytical grade. Tris–HCl buffer (10 mM, pH 7.0) was used as working medium. 2.1. Measurement of UV–vis spectra The bR preparation was studied by measuring the absorbance of bR on quartz plate/or on ATO electrode or in solution between 370 and 700 nm in absence and the presence of external light source using a double beam UV–vis spectrophotometer (Systronics, India, mode 2201). 2.2. Electrochemical measurement of immobilized bR on ATO electrode The electrochemical measurements were made using immobilized bR on ATO electrode as described earlier [57]. The electrochemical measurements were performed with a Solartron Electrochemical Interface (model 1287, Solartron, U.K.) connected to a PC through the serial port. The electrode response
was recorded and plotted with a Pinter. A one compartment home made cell with a working volume of 5 ml and a bR immobilized ATO electrode, platinum foil auxiliary electrode and Ag/AgCl reference electrodes were used for the measurements. The electrochemical cell as described earlier was used as such [58]. However, the light source (Fig. 1a and b) was re-designed to get relatively better stability. A device to expose the working electrode with external light source (Fig. 1a and b) was made which was attached to the back portion of transparent Perspex sheet onto which a working electrode was placed into the electrochemical cell with a three electrode configuration (Fig. 1c), with the arrangement of blue (Fig. 1a) and yellow (Fig. 1b) filters between working electrode and light source. The light source was a 50 W small light bulb placed within reflecting mirror designed in such a way that the transmitted light of an angle of 25◦ . The electrochemical measurements were made by polarizing the working electrode at desired potentials followed by the measurement of steady-state background current. Subsequently, the working electrode was exposed to light using a blue filter followed by a yellow filter. The photo-current as a function of time was recorded and plotted. The photo-current was measured at different potentials and with the light expose to the working electrode for sufficient duration to reach steady value followed by switching off the light exposure. At the optimum potential, the photo-current as a function of time was measured in the presence of varying concentrations of arginine and ammonium chloride. The experiments were performed in Tris–HCl buffer (10 mM, pH 7.0) and at 25 ◦ C.
Fig. 1. Home made photoelectrochemical cell and light source equipped with yellow and blue filters. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 3. (A) Spectra of bacteriorhodopsin in solution: curve 1 represents the absorption spectra in dark whereas curves 2, 3 and 4 represent the same in the presence of external yellow light [2 = control, 3 = in the presence of ammonium ion and 4 = in the presence of benzylamine]. (B) Absorption spectra of bacteriorhodopsin in solid-state. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) Fig. 2. Widely accepted photocycle of bacteriorhodopsin.
3. Results and discussion 3.1. Photosensitivity of bacteriorhodopsin The power of rhodopsin in image resolving eye is amazing and the technology behind that is perfect till the existing limitation of brain simulation. The knowledge available so far reveals that the generation of this perfect technology is basically due to integrated function of chromophore having close to 100% quantum efficiency together most efficient signal transduction in co-ordinated neural network justifying the existence of nanoelectronics in visual reception. Nevertheless, we have been trying to explore the possibility of generating molecular sensors and artificial retina based on optoelectrochemistry of bR within our resources. Fortunately, we have been able to record the sensitivity of bR photocycle on the presence of amine-residue and ammonium ion first time. The recordings of our observations were not concerned to fast kinetics of the bR photo cycle and basically concerns the observation recorded in finite time in dark and light. Before going ahead on this finding, it is now important to have a quick look of the photo cycle consisting of fast and slow kinetic part of bR as shown in Fig. 2. The cycle can be illustrated into six steps having major components of: (1) isomerization, (2) ion transport and (3) accessibility change. On photo-excitation of the chromophore, it first isomerizes from an all-trans to a 13cis configuration (step 1) followed by a proton transfer from the Schiff base to the proton acceptor Asp-85 (step-2). The proton transfer ultimately referred as to be de-protonation step (iontransfer step part-2) leads the formation of M-state. The Schiff base existed on M-state is then reprotonated from Asp-96 from the cytoplasmic surface and retinal reisomerizes thermally and the accessibility of the Schiff base switches back to extra cellular to reestablish the initial state (accessibility change, step-3). The
accessibility is switched from extra cellular to intracellular and thus the presentation of M-state requires inversion in intracellular to extra cellular direction. Fig. 2 justify the most accepted photo cycle of bR based on the data on fast and slow kinetics of the photocycle. We have tried to understand the slow part of the photo cycle as the existence of bR in two state might leads to the development of many molecular devices to be referred as practical molecular electronics materials as mentioned in the preceding sections. Accordingly, we proceeded to record the spectra of bR in light and dark to examine the stability of the same for practical applications. The results are recorded in Fig. 3A in dark (1), in the presence of external light (2), in the presence of external light and ammonium chloride (3) and in the presence of external light and benzylamine (4). The spectra were recorded in 10 mM phosphate buffer pH 7.0. The spectra clearly demonstrate the dependence of M-state on the presence of these chemical and opens the possibility for the exploitation of bR in the development of molecular sensors. In this direction, we further attempted to study the photochemistry of bR in solid-state on ATO electrode for its exploitation in opto-electrochemistry. Fig. 3B shows the spectra of bR in solid-state on ATO electrode and justify that suitability of the material in solid-state on ATO surface (Fig. 3B). The above preliminary finding directed our attention to investigate the photo activity of bR under following conditions: (i) on-line probing of bR at 410 nm under dark followed by illuminating the bR in external light and subsequently to record the decay kinetics at 410 nm after shutting down the external light source, (ii) on-line probing of bR at 530 nm under dark followed by illuminating the bR in external light and subsequently to record the decay kinetics at 530 nm after shutting down the external light source, (iii) on-line probing of bR at 640 nm under dark followed by illuminating the bR in external light and subsequently to record the decay kinetics at 640 nm after shutting
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Fig. 4. The kinetics of decay of the D96N mutant at 410, 530 and 640 nm (1) control, (2) in the presence of ammonium ion (0.003 M) and (3) in the presence of benzylamine (0.04 M) in 0.01 M phosphate buffer pH 7.0. The initial data recorded between 0 and 20 s (region i) represent the absorption in absence of external light data recorded between 20 and 40 s (region if) show the absorbance in the presence of external light whereas the rest of the curve (40–60 s; region iii) shows the absorption change in dark, back to the ground state (bR, 0.25 mg/ml).
down the external light source [47]. Such recordings are shown in Fig. 4. Interesting observations on slow kinetics of bR have been recorded and trigger the use of bR in molecular sensing based on kinetic data. These recordings have been evaluated in following three type of analysis namely (a) half time decay parameter at 410, 530 and 640 nm under control, in the presence of ammonium ion and in the presence of benzylamine, (b) total absorption at 410, 530 and 640 nm under control, in the presence of ammonium ion and in the presence of benzylamine and (c) absorption recorded in 500 ms at 410, 530 and 640 nm under control, in the presence of ammonium ion and in the presence of benzylamine. These findings are shown in Fig. 5. The
results again justify remarkable dependence of slow kinetics of bR photocycle on such chemicals. 3.2. Probing of enzymatic reaction The photocycle of bR has been susceptible to the presence of proton concentration even in wild type however become more apparent in D96N mutant bR. D96N mutant bacteriorhodopsin has been used in this communication which differs from the wild type by exchanging 96-residue of amino acid using a non-protonated residue. Such exchange perturbs the auto protonexchange ability of biomolecule that ultimately influences the
Fig. 5. Variation of the half time (T1/2 ), absorption of decay kinetics of decay kinetics and variation of the absorption of decay kinetics recorded at 410, 530 and 640 nm within the initial 500 ms recorded at 410, 530 and 640 nm of D96N mutant bR (0.25 mg/ml): (1) control, (2) in the presence of ammonium ion (0.003 M) and (3) in the presence of benzylamine (0.04 M) in 0.01 M phosphate buffer pH 7.0.
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Fig. 6. (A) The effect of enzymatic reaction kinetic of M-state decay of D96N bacteriorhodopsin at 410. The initial data recorded in 0–5 s represent the absorption in light whereas the rest of the decay curve shows absorption in dark back to ground state: 1 = control, 2 = in the presence of urease (0.3 mg/ml) and urea 0.02 M, 3 = in the presence of acetyl cholinesterase (0.08 mg/ml) and acetylcholine 0.02 M. (B) Variation in half time decay (T1/2 ) of D96N bacteriorhodopsin in the presence of increasing concentrations of benzylamine and ammonium ion (bR = 0.31 mg/ml).
kinetics of de-protonation and re-protonation processes of the photo cycle. Accordingly, the photochemistry of bR becomes dependent on the proton concentration present extra cellular side of bio-membrane. We have been able to record the probing of enzymatic reactions first time based on monitoring the slower kinetics of photo cycle [46]. The results as shown in Fig. 6 show the slower kinetic part (M-state decay) as a function of acetyl cholinesterase (AChE)- and urease-catalyzed reactions. The absorbance at 412 nm was recorded in the presence of varying concentrations of respective substrates [control (Fig. 6A, curve 1), urea (Fig. 6A, curve 2) and acetylcholine (Fig. 6A, curve 3). The measurement of decay kinetics at 412 nm was made. Again the results justify the dependence of photo cycle on the alternation proton concentration generated out side the extra cellular fluid. Urease catalyzed reaction generates base and thus causes reduction in proton outside extra cellular side whereas acetyl cholinesterase (AChE) catalyzed reaction generates acid thus increases the proton concentration under similar concentration. However, urease catalyzed reaction also produces NH4 + ion that really influences the photo cycle. Accordingly, the direct effect of pH variation could not be evaluated. The data in Fig. 6B shows inversion in kinetics of slow part of the photo cycle in the presence of benzylamine and ammonium chloride. The measurements were again conducted at 412 nm similar to that of recorded for Fig. 6A. 3.3. Photo-electrochemical investigations on bR photo cycle Since the bacteriorhodopsin is a highly sensitive photoactive material, the optical transparency of the immobilized matrix is one of the important requirements for practical applications. Apart from many matrices used so far, the matrix comparable to optical glass could be more suited as one of the option for practical applications. Accordingly, sol–gel glasses, com-
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parable to conventional optical material, made under specific conditions and derived from alkoxysilane precursors through sol–gel process is getting introduced not only for specific applications in studying bR photochemistry but to cover wide range of optical materials as well. Introduction of organic functionality in alkoxysilanes precursors, leading to organically modified sol–gel glass (ormosil) that might have additional advantage for anchoring targeted molecules and consequently making the process feasible to the production of functionalized optical materials for exploitation in many directions that has not been possible using conventional glasses so far. The ormosil derived from the functionalized alkoxysilane precursors at least trigger the introduction of such optically transparent material in studying the photochemistry of bacteriorhodopsin. Before providing details into the use of ormosil films in stabilizing the bR for opto-electrochemical investigations, we first attempted to study the photoelectrochemistry of bR present on ATO electrode and placed into electrochemical cell as shown in Fig. 1 or even as described earlier [58]. The results on photoelectrochemical responses are shown in Fig. 7. In this case, bR was simply adsorbed on ATO electrode and the membrane protein was not oriented. There is remarkable dependence of bipolar response of bR in dark and in the presence of light with significant dependence of photocurrent on applied potential. The home made light source as shown in Fig. 1 was used for exposure of bR on ATO electrode. The yellow light was allowed to pass though the working electrode for desired time. The recording as shown in Fig. 7A represents the data on photoelectrochemical response of un-oriented bR at −100 mV versus Ag/AgCl, Fig. 7B shows the same at 100 mV versus Ag/AgCl whereas Fig. 7C shows at −30 mV versus Ag/AgCl. On increasing the negative potential there is an increase in photocurrent whereas increase in positive potential reduces the magnitude of photocurrent. The recording shown in Fig. 7D and E justify the variation in bipolar behavior of photocurrent on altering the exposure of external light on bR. The external yellow light (Fig. 1) was exposed to bR sample for 10 s in case of Fig. 7D whereas the same was reduced to 1 s for Fig. 7E. There is transition from bipolar behavior when exposure time is reduced from 10 to 1 s. This preliminary observations although demonstrated in photo-sensing of targeted analyte (Fig. 8) however the finding provides an insight to use this molecule in designing artificial retina after suitably configuring the bR on optical material. After each set of photocurrent measurement, the bR was exposed to blue light. The data recorded in Fig. 8 shows the results on photocurrent at varying concentrations of the chemicals affecting the photo cycle. The electrode was polarized at −30 mV versus Ag/AgCl followed by exposure of the working electrode to external yellow light for 10 s. There is an increasing trend in both forward and backward photocurrent on increasing the lysine (Fig. 8a)/Arginine (Fig. 8b) concentration whereas the same shows decreasing trends when ammonium ions are present under similar conditions. It should be noted that in control (Fig. 8a) the response is bipolar however slightly less exposure time (manual error) might have caused diffused distinction of steady-state in control. Fig. 9 shows the typical recordings in the presence and absence of amine and ammonium ion by exposing the working
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Fig. 7. A typical recording of photocurrent at various applied potentials: (A) −100 mV, (B) +100 mV and (C) −30 mV vs. Ag/AgCl. The working electrode was exposed to yellow light for >12 s followed by recording in the dark as a function of time, (D) typical recording of the variation of photocurrent followed by exposure of working electrode for >12 s and subsequent recording in the dark as a function of time at −30 mV vs. Ag/AgCl. (E) A typical recording of the variation of photocurrent after exposure to light of working electrode for 1 s followed by subsequent recording in the dark as a function of time at −30 mV vs. Ag/AgCl. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
electrode for 1 s only. Even 1 s exposure time drastically altered the peak current in the presence of such analyte reflecting the possibility of exploitation of the bR in molecular photo-sensor designing. It appears that proton translocation sites linked to faster portion of photo cycle are kinetically influenced in the
Fig. 8. (a) A typical recording of the variation of photocurrent followed by exposure of working electrode for >12 s and subsequent recording in the dark as a function of time at −50 mV vs. Ag/AgCl in the absence (control) and presence of lysine (0.01, 0.02 and 0.03 M). (b) A typical recording of the variation of photocurrent followed by exposure of working electrode for >12 s and subsequent recording in the dark as a function of time at −50 mV vs. Ag/AgCl in the presence of arginine 0.05 M. The two insets show the result on control and in the presence of arginine (0.01 M).
presence of these amino- and ammonium residues, however, the mechanism of such interaction require thorough investigation and originate from the exceptional properties of proton dynamics which caused the existence of life on earth. These residues possibly affect the charge density near the retinal induced by its photo-isomerization followed by extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pKa values. The next stage of the investigation was concerns to coupling the bR within ormosil films and concerns to retain the photo activity of bR within isolated transparent matrixes. As a trial, we have worked on sandwiching bR using ormosil derived from phenyltrimethoxysilane, 3-aminopropyltrimethoxysilane and 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane. The preliminary results demonstrated interesting observations for practical usability of the material in technological development. The detailed output would be presented in a later publication.
Fig. 9. Typical recordings of photocurrent for the exposure of working electrode for 1 s at −30 mV vs. Ag/AgCl under various conditions: (a) control recording in the absence of amine and ammonium chloride, (b) in the presence of ammonium chloride and (c) in the presence of cysteine (0.015 M).
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4. Conclusion The present article demonstrates the science of bacteriorhodopsin which has potentiality in generating nano-devices with key references supported by the contributions made in our laboratory. The structure, habitat, properties of this biomolecule are discussed. The examples of nano-devices possibly emanating form bR have also been discussed. Attentions have been made on the development of bR-based nano-devices, i.e., molecular transistor, molecular motor, molecular gate, artificial retina and molecular sensors. Some important finding based on spectroscopy and photo-electrochemistry conducted in our laboratory has been summarized. The sensitivity of M-state to the presence of some amine and amino-compounds are reported based on spectroscopic and photo electrochemical observations. Both observations reveal that the presence of amine causes increase in life M-state whereas ammonium-residue affect in opposite fashion. The photoelectrochemical response of bR is found sensitive to the magnitude and polarity of applied voltage. In the presence of light, the biomolecule shows forward photocurrent whereas in absence of the same backward photocurrent is recorded. An increase in negative polarization potential result increase in forward and backward photocurrent. The bipolar response recorded on electrochemical polarization of bR molecule in the presence and absence of light and amine-/ammonium-residues has been reported reflecting its novelty in possible designing of artificial retina. The duration of light exposure time on photo-electrochemical bipolar response of bR are also reported that provide valuable information for technological exploitation of bR molecule in several other directions. Acknowledgement The author is thankful to DST, New Delhi for financial support. References [1] D. Osterhelt, W. Stoeckenius, Proc. Natl. Acad. Sci. U.S.A. 70 (1973) 2853. [2] D. Oesterhelt, W. Stoeckenius, Methods Enzymol. 31 (Pt. A) (1974) 667–678. [3] R. Henderson, P.N. Unwin, Nature 257 (1975) 28–32. [4] L.O. Essen, R. Siegert, W.D. Lehmann, D. Oesterhelt, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 11673–11678. [5] H. Belrhali, P. Nollert, A. Royant, C. Manzel, J.P. Rosenbusch, E.M. Landau, E. Pebay-Peyroula, Struct. Fold. Des. 7 (1999) 909–917. [6] M.B. Jackson, J.M. Sturtevant, Biochemistry 17 (1978) 911–991. [7] V.I. Shnvrov, P.L. Mateo, FEBS Lett. 324 (1993) 237–240. [8] K. Hiraki, T. Hamanaka, T. Mitsui, Y. Kito, Biochim. Biophys. Acta 647 (1981) 18–28. [9] M. Rehorek, M.P. Heyn, Biochemistry 18 (1979) 4977–4983. [10] G. Wald, P.K. Brown, Proc. Natl. Acad. Sci. U.S.A. 36 (1950) 84. [11] J. Nathans, D.S. Hogness, Proc. Natl. Acad. Sci. U.S.A. 81 (1984) 4851. [12] R.A. Bogomolni, R.A. Baker, R.H. Lozier, W. Stoeckenius, Biochim. Biophys. Acta 440 (1976) 68–88. [13] L. Lasse, F. Michael, P. Jussi, P. Sinikka, T. Jaaskelainen, Opt. Mater. 27 (2004) 57–62.
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