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Nanometer Arrays of Functional Light Harvesting Antenna Complexes byNanoimprint Lithography and Host-Guest InteractionsMaryana Escalante,†Yiping Zhao,‡,§Manon J. W. Ludden,‡Rolf Vermeij,†John D. Olsen,|Erwin Berenschot,§C. Neil Hunter,|Jurriaan Huskens,*,‡Vinod Subramaniam,*,†and Cees Otto*,†Biophysical Engineering Group, Molecular Nanofabrication Group, Transducer Science and Technology Group,and MESA+ Institute for Nanotechnology, UniVersity of Twente P.O. Box 217, 7500 AE Enschede, TheNetherlands, and Department of Molecular Biology and Biotechnology, UniVersity of Sheffield,Sheffield S10 2TN, U.K.Received April 17, 2008; E-mail: c.otto@utwente.nl; v.subramaniam@utwente.nl; j.huskens@utwente.nlSupramolecular interactions play a key role in the functionalarchitecture of nature. On patterned surfaces, interactions can beadjusted in strength and further modulated by the orientation oftarget molecules1,2Here, we have engineered functional orderedarrays of purified components of the photosynthetic system. Werelied on multivalent interactions to drive the selective assemblyof functional light harvesting LH2 antenna complex onto Nanometerstructured β-cyclodextrin (β-CD) monolayers2patterned by nanoim-print lithography (NIL).3,4The nanomachinery of the photosynthetic bacterium Rhodobactersphaeroides has been an invaluable model for the study ofbiophysics, biochemistry, and molecular biology of photosynthesis.5The membrane-bound LH2 complex is built of nine identicalsubunits each consisting of an R and a β polypeptide. A total of 27bacteriochlorophyll (BChl) molecules (18 BChl B850 and 9 B800)are bound to this structure having dimensions of ∼6 nm in heightand ∼6 nm in diameter.6,7LH2 are interesting candidates forapplications in synthetic light converting circuits because of theirwell-defined optical properties, such as a broad spectral range, highabsorption cross section, efficient energy transfer8and highphotostability. Photosynthetic antenna systems have been used instudies exploiting covalent9and electrostatic interactions10topromote attachment to a chemically defined surface. A majorchallenge remains in the control of the interfacial properties andthe associated multiple weak interactions to produce and optimizeorganized molecular structures with controlled directional energymigration.Here, we show an approach based on a combination of site-directed mutagenesis, NIL and multivalent host-guest interactions.LH2 complexes were engineered with cysteine residues at thepenultimate position of the C-terminus of the R polypeptide chain.These strategic positions at the periplasmic face ensured theorientation of all of the protein complexes upon binding to thesurface. The cysteine residues were modified with iodoacetyl-tri(ethylene glycol) mono(adamantyl ether), AdI, block 3, Chart 1.Protein aggregates in an aqueous buffered solution of 20 mMHEPES, pH 8.0, 0.03% n-dodecyl-β-D-maltoside (β-DDM) weremixed in 1:20 mol equiv with the AdI in 1.3% dimethyl sulfoxide(DSMO) to yield an average of three adamantyl molecules linkedto each protein complex, hereafter referred to as AdnLH2.11When adsorbing AdnLH2 onto a βCD-coated glass substrate(Chart 1, block 1), hexa(ethylene glycol)mono(adamantyl ether)AdHEG, block 2, served as a temporary blocking agent for theβ-CD cavities, preventing nonspecific adsorption by shielding thesurface with the HEG chain.12The monovalent AdHEG is latereffectively displaced through competition by exploiting the higheraffinity of the multivalent AdnLH2.12The assembly on the patternedsurface is depicted in block 4.We used a custom-built hybrid high resolution scanning probe-spectral microscope13to characterize the patterned proteins in liquidconditions. A fluorescence titration allowed us to simultaneouslyaddress the optical properties after modification of the LH2 complexand the specificity of the binding on nonpatterned β-CD surfaces.Upon excitation of the LH2 complexes via the B800 BChl (donor),the energy is then transferred within the complex to the B850(acceptor) and ultimately emitted as fluorescence. A dilute solutionof nonmodified LH2 complexes was incubated onto the β-CDmonolayer and rinsed with buffer. The average emission spectra(blue box trace, Figure 1a), indicates a high contribution ofnonspecific adsorption. Subsequently, after pretreating the surfacewith 1 mM solution of AdHEG, a solution of nonmodified LH2complexes in 1 mM AdHEG was incubated onto the substrate. Thenonspecific adsorption was reduced by 94% (open box trace). Theprevious experiment was repeated with AdnLH2 complexes. Theincrease in intensity of the emission signal (green star trace) revealsthat the protein complexes specifically bind to the surface byreplacement of the monovalent AdHEG with the multivalentAdnLH2. This replacement12and the observed stability againstrinsing with a βCD solution2are evidence for the formation of stablemultivalent complexes via at least 3 Ad linkers. Quantitatively, thespectral response from the immobilized AdnLH2 complexes showedno visible shift of the emission maximum, ∼868 nm, with respectto bulk measurements of nonmodified LH2 complexes (red triangletrace). This observation is compelling evidence that the completeprocedure of labeling and surface adsorption has maintained the†Biophysical Engineering Group, University of Twente.‡Molecular Nanofabrication Group, University of Twente.§Transducer Science and Technology Group, University of Twente.|University of Sheffield.Chart 1.Representation of Host, Guest, and Target Moleculesaa(1) β-CD heptamine, host molecule,2(2) Hexa(ethylene glycol)-mono(adamantyl ether) (AdHEG), (3) iodoacetyl-tri(ethylene glycol))mono(adamantyl ether), (AdI), (4) AdnLH2 on the β-CD monolayer.Published on Web 06/21/200810.1021/ja802843m CCC: $40.75 © 2008 American Chemical Society88929J. AM. CHEM. SOC. 2008, 130, 8892–8893