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Jun 3, 2010 - transfer this additionally collected energy to bR via Förster resonance energy transfer (FRET). ... energies (within the solar spectrum) and are able to transfer ..... by a homemade source based on a 650 W halogen lamp.

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Resonance Energy Transfer Improves the Biological Function of Bacteriorhodopsin within a Hybrid Material Built from Purple Membranes and Semiconductor Quantum Dots Aliaksandra Rakovich,† Alyona Sukhanova,‡,§ Nicolas Bouchonville,‡ Evgeniy Lukashev,| Vladimir Oleinikov,⊥ Mikhail Artemyev,# Vladimir Lesnyak,∇ Nikolai Gaponik,∇ Michael Molinari,‡ Michel Troyon,‡ Yury P. Rakovich,† John F. Donegan,† and Igor Nabiev*,‡,§,O †

School of Physics and CRANN, Trinity College Dublin, Dublin 2, Ireland, ‡ Universite´ de Reims Champagne-Ardenne, 51100 Reims, France, § CIC nanoGUNE Consolider, E-20018 Donostia-San Sebastian, Spain, | Department of Biophysics, Lomonosov Moscow State University, 119992 Moscow, Russian Federation, ⊥ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russian Federation, # Institute of Physico-Chemical Problems, Belarusian State University, Minsk, Belarus, ∇ Physical Chemistry, Technical University of Dresden, 01062 Dresden, Germany, and O IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain ABSTRACT Purple membrane (PM) from bacteria Halobacterium salinarum contains a photochromic protein bacteriorhodopsin (bR) arranged in a 2D hexagonal nanocrystalline lattice (Figure 1). Absorption of light by the protein-bound chromophore retinal results in pumping the protons through the PM creating an electrochemical gradient which is then used by the ATPases to energize the cellular processes.1 Energy conversion, photochromism, and photoelectrism are the inherent effects which are employed in many bR technical applications.2,3 bR, along with the other photosensitive proteins, is not able to deal with the excess energy of photons in UV and blue spectral region and utilizes less than 0.5% of the energy from the available incident solar light for its biological function.4 Here, we proceed with optimization of bR functions through the engineering of a “nanoconverter” of solar energy based on semiconductor quantum dots (QDs) tagged with the PM. These nanoconverters are able to harvest light from deep-UV to the visible region and to transfer this additionally collected energy to bR via Fo¨rster resonance energy transfer (FRET). We show that specific nanobio-optical and spatial coupling of QDs (donor) and bR retinal (acceptor) provide a means to achieve FRET with efficiency approaching 100%. We have finally demonstrated that the integration of QDs within PM significantly increases the efficiency of light-driven transmembrane proton pumping, which is the main bR biological function. This new QD-PM hybrid material will have numerous optoelectronic, photonic, and photovoltaic applications based on its energy conversion, photochromism, and photoelectrism properties. KEYWORDS Quantum dot, bacteriorhodopsin, FRET, hybrid material, photovoltaic

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urple membranes of the bacteria Halobacterium salinarum are unique natural membrane protein crystal patches where a single integral protein bR forms tight trimers arranged into an ultrastable lattice with a unit-cell dimension of 6.2 nm (Figure 1a,b). The photochromic and photoelectric properties of bR are coupled directly to its biological function.5 Upon absorption of light, bR undergoes a cyclic sequence of conformational transitions between the distinguishable photointermediate states accompanied by transport of protons from the cytoplasmic to the extracellular surface of the PM. The high quantum efficiency of the initial

bR state guarantees an efficient photoisomerization of the bR-linked chromophore (retinal) that is located near to the center of the PM, at a distance of nearly 2.5 nm from both PM surfaces (Figure 1).6 This distance is far less than the average Fo¨rster radius of 5 nm found in energy transfer processes.7 The proton transport within PM occurs with the initial charge separation and then the de- and reprotonation of the retinal.2 The intra- and extracellular surfaces of PM have different charges providing a permanent dipole moment across the PM.1 Evolution has often solved problems in nature similar to those that man attempts to solve in development of lightharvesting inorganic compounds. In this way bioelectronics and biophotonics have shown considerable promise. Much of the current research effort in this field is directed toward

* To whom correspondence should be addressed, [email protected] or [email protected] Received for review: 04/19/2010 Published on Web: 06/03/2010 © 2010 American Chemical Society

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FIGURE 1. | Structural organization of purple membrane-quantum dot hybrid material and optical properties of bacteriorhodopsin and quantum dots. All objects drawn in panels a and b are to scale. Photons are absorbed by the QD immobilized on the surface of the PM containing bR. Panel a shows that each bR molecule contains one chromophore with absorption in the visible region of optical spectrum (retinal, shown in purple). Retinal is located in the center of the PM, nearly 2.5 nm from each membrane side. An exciton from the QD (donor, shown in orange) is transferred via FRET to the chromophore of bR (retinal, acceptor) thus initiating retinal photoisomerization, charge separation near the retinal’s Schiff base, bR photocycle, and proton pumping through the PM. This energy transfer also results in the strong quenching of the PL of QD. Panel b shows QD complex with the “white membrane”sthe same bR-containing PMs as those shown in panel a but with the retinal carefully extracted. Here, QDs immobilized on the surface of the PM do not transfer an exciton to the acceptor because this acceptor (retinal) is absent. The QD remain fluorescent due to the electron-hole recombination within the QD. Panel c shows UV-vis spectra of PMs (purple line), white membranes (gray line), UV-vis spectrum for one of the types of QDs used in the study (filled with blue), and the PL emission spectrum of these QDs (black line). Panel d shows UV-vis absorption and PL emission spectra of some CdTe QD used in the study. Spectra of QD550 (green curves), QD600 (orange), and QD650 (red) are shown here.

harvesting system, it is able to utilize only 0.1-0.5% of the solar light. An understanding of the methods to enhance the light harvesting capability of bR provides a strong impetus to demonstrate bR-based device optimization (Figure 1). Monodispersed semiconductor photoluminescent (PL) CdTe14 or CdSe/ZnS15 QD have exceptionally high extinction coefficients (Figure 1c,d). QD are ultrastable against photobleaching, and the quantum confinement effect yields PL emission energy that varies as a function of the size of the QD. The spectral width and position of the optical bands in the QD can be tailored by controlling their size, while surface chemistry permits adjustment of their surface functionalities.16-18 In this study, we utilize highly PL QDs as the energy converters that absorb light in a wide range of photon energies (within the solar spectrum) and are able to transfer the harvested energy to the bR chromophore retinal with high efficiency (Figure 1a,c). In our experiments, the natural

self-assembled monolayers of protein-based photonic devices.8 Although a number of proteins have been explored for bioelectronics device applications,9,10 bR has received the most attention.3 The substantial number of publications and patents filed is a strong indicator that the use of bR in technical applications is a technological reality.3 One of the main domains for effective competition of bR-based devices with existing technologies is the field of dynamic applications, where thousands of cycles between two or more distinguishable and reversible states are required.2 Nanotechnology may open the way to enhance the performance of bR biological function. Photosensitive proteins are not able to interact with UV photons and do not absorb them at all; if they are absorbed, they might destroy the light-harvesting chromophores11 or induce apoptotic-like cell death.12 Owing to light-harvesting system, the energy harvesting in plants has a maximum efficiency of 5%,4,13 whereas in bR, which does not have any specific light© 2010 American Chemical Society

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groups are also exposed.21 This means that the cytoplasmic surface of PM contains a sufficient number of positively charged groups which are localized exactly in the positions of the bR trimers. This fact explains why, at relatively low QD/bR ratios, the negatively charged QD may interact specifically with the bR protein trimers on the surface of the PM rather than with the negatively charged surface of the lipids (Figure 2). Panels a and b of Figure 3 show that assembling of CdTe QDs of different diameters (colors) and PMs leads to efficient quenching of exciton emission of QDs which suggests that an efficient FRET from QD to bR is occurring. Endorsement of FRET as the principle QD quenching mechanism was obtained from important data on the comparative quenching of QDs with the PMs and WMs (WMs are exactly the same membranes as the PMs, but they do not contain retinal that acts as the FRET-acceptor in QD-PM complexes). Figure 3a shows that the quenching of QDs by WMs is 4-5 times less than that with the PMs. This experiment provided us with a quantitative level of the possible contribution of nonradiative QD quenching in the PM or WM hybrids and proved that the main mechanism of QD quenching with PM is FRET. Figure 3b shows the difference in the quenching of CdTe QDs of two different colors (diameters) with the PMs. The smaller QD590 were quenched more efficiently at same bR to QD molar ratios, when compared to the quenching of QD650 PL. Such difference in quenching behavior is expected when one considers the smaller donor-acceptor distance between QD and the retinal of the bR protein, as well as the better spectral overlap between the PL band of QD590 with the absorption spectrum of bR’s ground state. It should also be mentioned that quenching of CdSe/ ZnS QD, which were solubilized and stabilized in aqueous solutions with PEG-based polymers or with low-molecularweight amino acid cysteine, was always much weaker than that for CdTe QD (Figure S5 in Supporting Information). We attribute this fact to larger diameters of CdSe/ ZnS QDs relative to CdTe QDs due to the thickness of the additional ZnS shell and of an additional organic shell created during the CdSe/ZnS QD water-solubilization (Table S1 in Supporting Information) that results in reduced FRET. In conjunction with the steady-state PL studies, important time-resolved PL data provided further evidence for FRET within the bR-QD hybrid material (Figure 3c,d). For example, the average lifetime for a complex consisting of ∼0.5 bR per CdTe QD650 was found to be decreased from 10 to 8 ns, which constitutes a decrease of about ∼20% (Table S2 in Supporting Information). This decrease in the average radiative lifetime of QD PL is expected if FRET exists,7 since the energy transfer from QD to the retinal provides an additional kinetic pathway for the decay of QD excitation.22 It is noteworthy that the integrated PL intensity for this complex decreased by ∼19% (Figure 3b), so there is very good agreement between the two measurements. Similar correla-

PM purified from bacteria Halobacterium salinarum was assembled with CdTe or CdSe/ZnS QD (Figure 1a). In order to develop an efficient hybrid material operating in the FRET regime, we carefully selected the PL colors (diameters) of the QD (donors of energy) to be optically coupled (spectrally overlapped) with the retinal (acceptor)sthe only chromophore of the PM that absorbs light in the visible region. Figure 1c shows that properly designed QDs dominate over the intrinsic absorption of the bR in the range from 400 to 600 nm by orders of magnitude. A description of the appropriate surface functionalization of QD and the procedure for their assembly with PM for the preparation of the hybrid material are presented in the Supporting Information. The retinal linkage with the bR protein (Schiff base) may be reduced under illumination and retinal may be carefully extracted from the PM according to a well-established protocol thus providing so-called “white membranes” (Figure 1b).19 White membranes (WMs) present an excellent control material for studying FRET between QDs and bR. The structure and morphology of the protein and lipid components in WMs are the same as those for PMs (see Supporting Information), providing equal possibilities for any nonradiative quenching of QDs upon their binding to either PMs or WMs, whereas the absence of retinal (acceptor) completely excludes the FRET-channel of QD quenching in their complex with WMs (Figure 1a,b). Figure 2a shows high-quality AFM images of bR within its natural PM. Here, one may see that the bR protein trimers are arranged in the clearly visible hexagonal lattice with a unit-cell dimension of 6.2 nm in good agreement with previously reported value.20 Importantly, one may see that the 2D organization of QDs assembled with the PM at an approximate 1:2 QD/bR molar ratio followed by washing out nonbound QDs is very similar to that of the bR trimers (Figure 2a,b). AFM profiles scanned along the PM surface and along the surface of PM-QD assemblies show that the period of bR trimer organization within the PM (6.2 nm) corresponds well to the period of QD organization on the PM upon assembling and purification of the hybrid material (6.09 nm). The data suggest specific binding of QDs with the bR trimers on the surface of the PMs at low QD/bR ratios. An increase of the QD/bR ratio in the QD-PM hybrids leads to the disappearance of the regular ordering of QDs on the surface of the PMs and formation of irregular multilayers. Supplementary Figure S6 (Supporting Information) shows the complexes of QDs with PMs at a 3:1 QD/bR molar ratio were such multilayers are formed. It should be mentioned that the PM contains 19 lipid molecules per one bR molecule.21 So, the average surface charge of the PM is negative as is determined by the charge of the heads of the lipid PO3- groups. The bR protein itself contains 14 basic lysine and argenine positively charged amino acid residues. Moreover, the cytoplasmic surface of the bR molecule contains nine exposed positive groups whereas on the exterior surface of the PM, three positive bR © 2010 American Chemical Society

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FIGURE 2. | Atomic force microscopy images of bacteriorhodopsin within its natural purple membrane (a, c) and the same images for the assemblies of purple membrane with quantum dots (b, d). Bottom parts of panels a-d show AFM profiles scanned along the blue lines indicated on the AFM images in the top parts. These profiles show that the period of bR trimer organization within the PM (6.2 nm, images of panels a and c) corresponds well to the period of QD organization on the PM upon assembling of the hybrid material (6.09 nm, panels b and d). The data show specific binding of QD with the bR trimers on the surface of the PM.

tions, although for much less efficient PL quenching and lifetime variation, were also observed for the PM complexes with the CdSe/ZnS QD (Figure 3d and Table S3 and Figure © 2010 American Chemical Society

S5 in Supporting Information). All these data confirm that the main mechanism of the quenching of QD PL with PM is indeed FRET. 2643

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FIGURE 3. Integrated photoluminescence and time-resolved photoluminescence decay as a function of bacteriorhodopsin to quantum dot molar ratios. Panel a shows variation of integrated PL of CdTe QD590 (λem ) 590 nm) as a function of concentrations of purple membranes (black curve) or white membranes (red curve). The concentration of QD was fixed at 1.4 µM and dilution factor and internal filter effects were taken into account as described in the Supporting Information. The optical densities in these experiments were always around Abs ) 0.07-0.08. (b) Variation of integrated PL for CdTe QD590 (blue triangle) and QD650 (purple circle) as a function of bR-to-QD molar ratios in the hybrid material. Experimental conditions were the same as in panel a. Panel c presents the time-resolved PL measurements for complexes of PMs with CdTe QD650 and panel d shows these experiments for complexes of PMs with CdSe/ZnS QD570. The changes in the decay of the PL can also be distinguished through changes in the slopes of the curves (especially at shorter times). In all cases the decays where biexponential; this is typical for measurements with colloidal QD.

provokes a photoinduced decrease of the concentration of protons (increase of pH) in a suspension of proteoliposomes. Figure 5 shows that the photoinduced pH-response detected in the suspension of our proteoliposomes is reversible and the recorded value of the variation of pH at 2 kW/m2 illumination is typical for the preparation of extremely highly oriented bR-containing proteolipisomes.24 Moreover, switching off the illumination leads to the complete relaxation of the system to the initial pH value. Figure 5 also shows the result of experiments with hybrid material made by immobilization of semiconductor QD on the surface of the proteoliposomes. Integration of CdTe QD590 within the PM even at the 1:5 QD/bR molar ratio induces a pronounced (>25%)increaseofthephotoresponseforbRprotonpumping. It is important to note that the thickness of the organic shell covering the QD fluorescent core is essential both for

We have in addition carried out the first proof-of-principle evidence of the increase of the efficiency of the biological function of bR in the presence of QDs. This demonstration was done using oriented proteoliposomes (Figure 4)slipid vesicles (liposomes) with the bR protein molecules incorporated within the walls of these vesicles in a highly oriented manner.23 Proteoliposomes with an average diameter of 240 nm were formed according to the classical protocol of Racker and Stoeckenius.23 Such proteoliposomes have a surface density of 0.06 nm-2, which corresponds to the density of hexagonal bR lattice package in native PM. In contrast to the bacterial PM, where bR pumps protons out from the cell,24 orientation of bR in the proteoliposomes prepared according to the protocol of Racker and Stoeckenius is known to be opposite so that the protons are pumped inside the proteoliposomes.24 As a result, photoillumination © 2010 American Chemical Society

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S5 in Supporting Information), as well as with the FRET efficiency of ∼18%, obtained from PL time-resolved data (Table S3 in Supporting Information). The photoresponse of bR within the hybrid material increases rapidly upon an increase of the power density of the photoillumination and then saturates at around 2 kW/m2 (Figure S7 in Supporting Information). It should be mentioned that the increase of the photoresponse due to the presence of QDs is found to be maximal at the biggest power densities and dependent on the quantity of QDs incorporated within the proteoliposomes. Assembling QDs and PMs in quantities exceeding 1:5 QD/bR molar ratios leads to apparent decrease in the efficiency of bR proton pumping (Figure S7 in Supporting Information). This may be explained by the internal filter effect due to the presence of a significant quantity of QDs forming multilayers or nonbound nanocrystals present in suspension. All these QDs would not participate in FRET with bR but would in fact block photoexcitation of bR. Additionally, we have to take into account that the photocycle of bR is a complicated process where the molecules proceed in a sequence of photodistinguishable conformational transitions. This process may be influenced by additional illumination with the wavelengths corresponding to some particular photointermediates. In this work we have demonstrated the effect of QD just on the bR ground state and this effect leads to an increase of the efficiency of the principle biological function of bRstransmembrane pumping of the proton. Use of QDs of different and carefully selected colors may specifically affect some selected intermediates of the bR photocycle thus permitting development of more complex hybrids with metastable or stable states and paving the way to development of novel optoelectronic switches or memory elements. In conclusion, we have demonstrated that QDs specifically immobilized on the surface of the purple membrane are able to play the role of a built-in light energy convertor by harvesting light which would not be absorbed efficiently by the purple membranes alone (from UV to blue region). Membrane-immobilized QDs were further demonstrated to be able to transfer the harvested energy via highly efficient FRET to this complex biological system. We have finally demonstrated a first proof-of-the-principle evidence that the bR being a part of engineered QD-PM hybrid material is able to utilize the transferred by QD additional energy to improve the efficiency of its biological function. Future work on optimization of hybrid material efficiency includes development of highly oriented PM-QD films with enhanced photovoltaic and optical switching properties. In such films, the internal filter effect should be decreased and higher efficiency should be achieved at the higher QD/bR ratios. In addition, CdTe QDs, used in our study, although demonstrating very efficient FRET, have moderate extinction coefficients. Further optimization of QD-PM hybrid material can be done using QDs with stronger absorptions

FIGURE 4. Organization and functionality of a complex composed from the liposomes containing highly oriented bacteriorhodopsin (proteoliposomes) and QDs immobilized on the surface of the proteoliposomes. All objects drawn in this figure are given to scale. (a) Insert on the right shows a fragment of the liposome’s membrane where bR is shown in blue and retinal is shown in red. Excitation is with white light. Illumination with photons of the orange color in the visible spectrum provides direct excitation of bR. Photons of higher energy (blue region) excite the QD only and they transfer their energy to bR via FRET. Absorption of photons by bR induces transfer of the proton inside the proteoliposome and the photoinduced increase of pH in the solution exterior of proteoliposomes which serves as a measure of the efficiency of the bR function. (b) Bacteriorhodopsin is organized into the trimers in the membrane of the proteoliposomes. The dimension of these trimers is very similar to the diameter of QDs used in the experiments on energy transfer.

the efficiency of FRET and for the increase of the biological function of bR. So, efficiency of FRET and an increase of photoresponse were found to be a maximum for CdTe QD covered with a stabilizing TGA shell of thickness

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