Selective Recruitment of Membrane Protein Complexes onto Gold

Dip-pen nanolithography (DPN) is employed to develop a generic array platform for the selective recruitment of membrane protein complexes. An atomic f...
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Selective Recruitment of Membrane Protein Complexes onto Gold Substrates Patterned by Dip-Pen Nanolithography Ramujnas Valiokas,*,§,† Sˇ arujnas Vaitekonis,† Goran Klenkar,§ Gediminas Trinkujnas,† and Bo Liedberg§ DiVision of Molecular Physics, Department of Physics, Chemistry and Biology, Linko¨ping UniVersity, S-581 83 Linko¨ping, Sweden, and Molecular Compounds Physics Laboratory, Institute of Physics, SaVanoriu 231, LT-02300 Vilnius, Lithuania ReceiVed December 13, 2005. In Final Form: February 16, 2006 Dip-pen nanolithography (DPN) is employed to develop a generic array platform for the selective recruitment of membrane protein complexes. An atomic force microscope tip inked with HS(CH2)16NH2 is used to generate aminoterminated domains on gold. These domains can be arranged into microscopic and submicroscopic patterns, and the untreated gold substrate is subsequently blocked with HS(CH2)2CONH(CH2CH2O)15CH3, a compound known to resist the unspecific binding of proteins and cells. The patterned gold substrate is exposed to an enriched membrane fraction from mutant Rhodobacter sphaeroides, which contains photosynthetic core complexes consisting of the reaction center and the light-harvesting complex LH1. The selective recruitment to the patterned domains, governed primarily by electrostatic interactions, is confirmed by contact mode atomic force microscopy.

Introduction A successful combination of molecular self-assembly and scanning probe manipulation has recently led to the development of new powerful methods for the direct generation of organic structures on solid surfaces (recently reviewed by Kra¨mer et al.1). These emerging techniques are regarded as promising tools for micro- and nanofabrication, and they are believed to have tremendous potential in the rapidly growing fields of proteomics and nanobiotechnology. For example, two novel techniques, dubbed tip displacement nanolithography2 and dip-pen nanolithography (DPN),3,4 have been employed for the immobilization of biological materials, such as DNA,5 antibodies,6,7 enzymes,7-9 viral particles,10,11 and lipid vesicles,12 as well as for nanoscopic control of cell adhesion.7 However, the applications of DPN and other scanning probe lithographic techniques in the field of membrane protein research have not yet been explored. The fundamental property of the membrane proteins is the strong dependence of their functional activity on the surrounding lipid * Corresponding author. E-mail: [email protected]. Phone: +370 5 2661640. Fax: +370 5 2602317. Address: Molecular Compounds Physics Laboratory, Institute of Physics, Savanoriu 231, LT-02300 Vilnius, Lithuania. § Linko ¨ ping University. † Institute of Physics. (1) Kramer, S.; Fuierer, R. R.; Gorman, C. B. Chem. ReV. 2003, 103, 43674418. (2) Xu, S.; Liu, G. Y. Langmuir 1997, 13, 127-129. (3) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S. H.; Mirkin, C. A. Science 1999, 283, 661-663. (4) Ginger, D. S.; Zhang, H.; Mirkin, C. A. Angew. Chem., Int. Ed. 2004, 43, 30-45. (5) Demers, L. M.; Ginger, D. S.; Park, S. J.; Li, Z.; Chung, S. W.; Mirkin, C. A. Science 2002, 296, 1836-1838. (6) Wadu-Mesthrige, K.; Xu, S.; Amro, N. A.; Liu, G. Y. Langmuir 1999, 15, 8580-8583. (7) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702-1705. (8) Wadu-Mesthrige, K.; Amro, N. A.; Garno, J. C.; Xu, S.; Liu, G. Y. Biophys. J. 2001, 80, 1891-1899. (9) Zhou, D. J.; Wang, X. Z.; Birch, L.; Rayment, T.; Abell, C. Langmuir 2003, 19, 10557-10562. (10) Cheung, C. L.; Camarero, J. A.; Woods, B. W.; Lin, T. W.; Johnson, J. E.; De Yoreo, J. J. J. Am. Chem. Soc. 2003, 125, 6848-6849. (11) Smith, J. C.; Lee, K. B.; Wang, Q.; Finn, M. G.; Johnson, J. E.; Mrksich, M.; Mirkin, C. A. Nano Lett. 2003, 3, 883-886. (12) Schonherr, H.; Rozkiewicz, D. I.; Vancso, G. J. Langmuir 2004, 20, 7308-7312.

environment. Therefore, the fabrication and manipulation of surface-supported membrane protein assemblies remains a challenge. In the present work, we address this issue by investigating the interaction of isolated cell membrane fragments with nanolithographically patterned amino-terminated self-assembled monolayer (SAM) domains10,13 separated by a poly(ethylene glycol) (PEG)blocking layer assembled from solution. As a relevant biochemical model system, we have chosen enriched membranes of mutant Rhodobacter sphaeroides with photosynthetic core complexes (so-called photosynthetic units (PSUs)) that consist of the lightharvesting complex (LH1) and the reaction center (RC).14 This is a crude and complex protein-lipid mixture containing the proteins of interest. It should be emphasized, however, that the relative PSU protein concentration in such a mixture is much lower than that in samples typically used for obtaining highresolution atomic force microscopy (AFM) images of twodimensional (2D) crystalline-like membrane protein assemblies.15,16 We discuss herein critical issues regarding the preparation and characterization of amino patterns on gold using AFM. We also demonstrate the practical possibilities of using site-directed self-assembly via DPN to achieve ultrahigh array element densities for membrane protein analyses and to construct new proteinbased architectures for future bioelectronic and bioanalytical devices. Experimental Section As substrates for DPN, we used 120 Å thick gold films that were electron beam deposited on top of a 10 Å titanium adhesion layer on chips cut from standard (100)-silicon wafers. For convenience, the chips were marked with a diamond needle using a wafer scriber (Karl Su¨ss KG, Germany). The electron beam evaporation of the metals was done in a Balzers UMS 500 P system operating at a base (13) Zhang, H.; Lee, K. B.; Li, Z.; Mirkin, C. A. Nanotechnology 2003, 14, 1113-1117. (14) Walz, T.; Jamieson, S. J.; Bowers, C. M.; Bullough, P. A.; Hunter, C. N. J. Mol. Biol. 1998, 282, 833-845. (15) Scheuring, S.; Seguin, J.; Marco, S.; Levy, D.; Robert, B.; Rigaud, J. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1690-1693. (16) Siebert, C. A.; Qian, P.; Fotiadis, D.; Engel, A.; Hunter, C. N.; Bullough, P. A. EMBO J. 2004, 23, 690-700.

10.1021/la053367w CCC: $33.50 © 2006 American Chemical Society Published on Web 03/09/2006

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pressure on the low 10-9 mbar scale. The evaporation pressure for Au was below 1× 10-7 mbar, and the evaporation rates were kept constant at 1 or 5 Å/s for titanium and gold, respectively. Prior to DPN, the gold substrates were cleaned in a 5:1:1 mixture of deionized (MilliQ, Millipore) water, 25% hydrogen peroxide, and 30% ammonia for 5 min at 80 °C (SC-1 solution), followed by rinsing in deionized water. The ink for the DPN process was a 200 µM solution of HS(CH2)16NH2 (a generous gift from Dr. Johan Ekeroth). The compound was found to dissolve partially in methanol. The Si3N4 cantilevers with a spring constant of k ) 2 N/m (MicroMasch, Spain) were gently washed in the SC-1 solution, rinsed in ethanol, and placed into a glass vial with the ink. After 5 min they were removed from the vial and dried under a gentle stream of N2 gas. The precleaned gold substrates were kept in water. Immediately before the experiment they were again rinsed in ethanol and finally blown dry with the N2 gas. DPN and topographic characterization were carried out using a multimode NanoScope IIIa (Digital Instruments, Santa Barbara, CA) with a 100 µm J-scanner. For the deposition of the NH2 thiol, a square of the desired size was scanned at a rate of 1 Hz with a resolution of 128 × 128 pixels. The patterned area was subsequently inspected in contact mode at a scanning rate of 5 Hz. The decorated substrate was removed from the AFM sample holder and immersed into an ethanolic solution of HS(CH2)2CONH(CH2CH2O)nCH3, n ≈ 15 (hereafter referred to as HSPEG750), which was purchased from RAPP Polymere Gmbh, Tu¨bingen, Germany, and used as received. The concentrations were 40 and 200 µM, and the adsorption time varied from a few hours to 24 h. The patterned and blocked samples were then washed in ethanol using an ultrasonic bath for 3 min. Those used for topographic characterization were blown dry with the N2 gas and immediately analyzed by AFM. The substrates used for the protein adsorption experiments were again sonicated for another 3 min in MilliQ water and then preconditioned by the deposition of a drop of the adsorption buffer (10mM Tris‚HCl, 150 mM KCl, 25mM MgCl2, pH 7.5) on the patterned area. An enriched membrane fraction from mutants of the photosynthetic purple bacteria Rhodobacter sphaeroides was a generous gift from Dr. Zˇ ivile˘ Katiliene˘ and Prof. Neal W. Woodbury, Arizona State University. The details of obtaining the fraction with chromatophores lacking the peripheral light-harvesting complexes LH2 are published elsewhere.17 Briefly, the bacteria were grown semiaerobically in a rich medium, harvested, and resuspended in 10 mM Tris‚HCl (pH 8). After sonication (3 times, 3 min each), the cell extracts were incubated with DNase and then centrifuged at a low speed (8000 rpm, 30 min, Sorvall GS3 rotor) to remove whole cells and debris. The membranes were isolated by high-speed centrifugation (45 000 rpm, 2 h 15 min, Beckman Ti45 rotor) and resuspension of the pellet by homogenization in 15 mM Tris‚HCl (pH 8), 1 mM EDTA, and 0.1 M NaCl. The chromatophore-containing membrane suspension was stored at -75 °C in 15% (v/v) glycerol. Typically, 50 µL of the adsorption buffer was placed on the patterned area of the chip, and 5 µL of the membrane suspension was subsequently injected into the drop. The membrane protein complexes were allowed to adsorb for 1 h, and the excess material was removed by gently rinsing the sample with an “imaging buffer”, 10 mM Tris‚HCl, 50 mM KCl, pH 7.5. The sample was always kept under the buffer and carefully inserted into the liquid cell on the AFM stage without exposing it to air. The characterization of the deposited membrane suspension was carried out using Si3N4 tips with a spring constant of k ) 0.1 N/m; (MicroMasch, Spain). The AFM was operated at a tip force of 0.5 nN, the scanning frequency was 5 Hz, and the resolution was 256 × 256 pixels. The images were analyzed using a commercial Nanoscope IV and a home-written software package.

Results and Discussion Nanolithographic Patterning of Amino-Terminated Thiols. The efficiency of the molecular transport of the ink molecules, HSC16NH2, from the AFM tip to the gold substrate was first (17) Freiberg, A.; Allen, J. P.; Williams, J. C.; Woodbury, N. W. Photosynth. Res. 1996, 48, 309-319.

Figure 1. DPN on polycrystalline gold using HSC16NH2 as ink. (a) A rectangular feature was generated by scanning the surface with an AFM tip loaded with the ink, and the topography of the processed area was obtained in contact mode in air immediately after the DPN process. (b) Section analysis along the white line in panel a. (c) The smallest features, dots, were generated by bringing the inked AFM tip into contact with the surface for around 1 s and subsequently imaging as described for panel a. (d) Section analysis along the white line in panel c.

assessed by scanning 1 × 1 µm2 domains in contact mode. Subsequent AFM analysis confirmed the presence of regular features with sharp edges and corners (Figure 1). The measured height of these features varied from 1.6 to 2.3 nm, values that are in good agreement with the thickness of a monolayer of HSC16NH2 molecules oriented perpendicularly to a gold surface. A good reproducibility could be achieved under room conditions for 1 × 1 µm2 patterns, that is, without precise control of the humidity. However, smaller features such as 500 nm-sized squares were harder to reproduce. The ultimate resolution of the patterning process was tested by bringing the AFM tip in contact with the gold surface for about 1 s. This resulted in the formation of spots with an average feature size of around 300 nm (Figure 1c). The dots could easily be reproduced and aligned with controlled separations, as shown in Figure 1c, and, most importantly, they still had the distinctive height of about 2 nm, which suggests a surface coverage (packing)

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similar to that for the larger square-shaped features achieved by scanning. It is known from earlier works that the resolution of the alkylthiolate features generated by DPN on polycrystalline gold can be well below 50 nm.4 Cheung et al. achieved a 50 nm line width by DPN using a complex, amino-terminated thiol compound, which contained alkyl, oligo(ethylene glycol), and amide moieties;10 although, these authors did not report on the topography of the generated DPN features. Several groups have shown, however, that factors such as the temperature, relative humidity of the ambient, and the treatment of the AFM tip could affect the molecular transport from the AFM tip to the substrate and thereby the resolution.18-22 Mirkin and co-workers experienced difficulties in transferring short chain aminothiols, such as cysteamine, using Si3N4 AFM tips.13 These authors decided instead to generate nanoscopic amino patterns by the solution self-assembly of cysteamine on gold nanoparticle arrays. In contrast, our results indicate that the ink transport properties using the HSC16NH2 molecule for direct molecular writing were not very different from those seen for methyl- or carboxyterminated long-chain alkylthiols,3,21 even without proper control of the ambient conditions, such as temperature and relative humidity. The patterned samples were then removed from the AFM sample holder and incubated in the solution of HSPEG750 for the passivation of the unmodified gold areas against unspecific binding of proteins.23,24 Subsequent inspection of the processed surface by contact mode AFM using the imaging buffer revealed an inverted height profile (Figure 2). The previously observed protrusions were transformed into depressions, which were 1.51.9 nm deep. Note that the ellipsometric thickness of an HSPEG750 SAM formed in solution and measured in air is 5.2 nm.25 On the other hand, recent AFM studies indicate that the thickness of equilibrated HSPEG750 SAM islands measured under buffer conditions is about 3.5 nm.26 Thus, the measured depth of the obtained array elements is in agreement with the height difference between the HSPEG750 and NH2 SAMs. This experiment also shows that the (sub)microscopic aminoterminated features remain stable against the self-assembly of the HSPEG750-blocking molecules, that is, no replacement of the shorter aminothiol seems to occur upon exposure to HSPEG750. Selective Adsorption of the Membrane Protein Suspension. The subsequent exposure of the above-discussed patterns to the enriched membrane fraction diluted in buffer resulted in the selective adsorption of the biological material to the aminogroup-containing areas (Figure 3). The section analysis of this typical image revealed that the depressions, which were due to the height difference between the NH2 and HSPEG750 SAMs, were efficiently filled and that the regular (square) shape of the original patterns was satisfactorily retained. The scanned area shown in Figure 3 shows that the thickness of the membrane fragments deposited on the NH2 SAM domains was at least 4 (18) Schwartz, P. V. Langmuir 2002, 18, 4041-4046. (19) Weeks, B. L.; Noy, A.; Miller, A. E.; De Yoreo, J. J. Phys. ReV. Lett. 2002, 88, 255505. (20) Sheehan, P. E.; Whitman, L. J. Phys. ReV. Lett. 2002, 88, 156104. (21) Rozhok, S.; Piner, R.; Mirkin, C. A. J. Phys. Chem. B 2003, 107, 751757. (22) Peterson, E. J.; Weeks, B. L.; De Yoreo, J. J.; Schwartz, P. V. J. Phys. Chem. B 2004, 108, 15206-15210. (23) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-436. (24) Benesch, J.; Svedhem, S.; Svensson, S. C. T.; Valiokas, R.; Liedberg, B.; Tengvall, P. J. Biomater. Sci., Polym. Ed. 2001, 12, 581-597. (25) Malysheva, L.; Klymenko, Y.; Onipko, A.; Valiokas, R.; Liedberg, B. Chem. Phys. Lett. 2003, 370, 451-459. (26) Rundqvist, J.; Hoh, J. H.; Haviland, D. B. Langmuir, 2005, 21, 29812987.

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Figure 2. Contact mode AFM images of arrays of submicroscopic features in buffer. Squares (a) and dots (c) were generated as described in Figure 1, and the unprotected gold areas were backfilled with HSPEG750. The array elements consisting of HSC16NH2 appear as dark features. The section analyses in panels b and d are along the white lines in panels a and c, respectively.

nm with respect to the bottom of the “well”. Also, the array elements contained a few hundred nanometers-sized patches with a typical height close to 10 nm. We also observed larger aggregates protruding about 20 nm above the surface, and they were difficult to image in contact mode (not shown). This suggests that the deposited membrane fragments had no uniform orientation and that they were rather inhomogeneous; that is, they consisted of different protein-lipid aggregates. A better control of the composition of the loading solution would certainly help to improve the resolution of the AFM images. Recent AFM studies revealed that a high concentration and purity of the proteins is required to obtain high-resolution images of analogous photosynthetic complexes.15,16,27 Thus, a precise structural charac(27) Bahatyrova, S.; Frese, R. N.; Siebert, C. A.; Olsen, J. D.; van der Werf, K. O.; van Grondelle, R.; Niederman, R. A.; Bullough, P. A.; Otto, C.; Hunter, C. N. Nature 2004, 430, 1058-1062.

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Figure 3. Selective immobilization of an enriched membrane fraction from Rhodobacter sphaeroides, which contains the light-harvesting complex LH1 and the RC, onto the array elements with surface amino groups. (a) The sample was imaged in a liquid cell under buffer conditions in contact mode. (b) The section analysis of the surface structures along the white line in panel a.

terization of the patterned membranes was unsuccessful, most likely because of a too low protein concentration in the suspension used. Nevertheless, the available resolution was sufficient to demonstrate that the membrane patches selectively attach to the array spots and that they are repelled by the Au-SPEG750 domains. The efficiency of reducing the unspecific binding by PEG can be estimated, for example, by surface roughness analysis. Before the exposure to the membranes, the root-mean-square (RMS) roughness of the PEG-terminated domains was 0.46 nm. After the exposure, the RMS only slightly changed to 0.31 nm. This is in sharp contrast to the height of the deposited features found within the array elements, which was in the range of a several nanometers. Indeed, it is known from the previous AFM studies on membranes from photosynthetic bacteria that the thickness of oriented PSUs containing membrane patches is close to 12 nm, and the typical height of subunits protruding from the membrane falls in the range 1.1-1.8 nm.15,27 The selective adsorption of the enriched membrane fraction to the array elements is primarily governed by the electrostatic interaction between negatively charged membrane components and the terminal amino groups of the SAMs that possess positive charges at the pH used.9,28-30 The neutral and hydrogen-bondaccepting Au-SPEG750 areas, on the other hand, exhibit resistance against the unspecific binding of the protein complexes. The objective of this work was to demonstrate the use of DPN for patterning membrane protein samples via generating arrays of amino-terminated compounds. In the present study, we do not perform detailed functional tests of the immobilized PSUs, nor do we focus on high resolution imaging of the protein complexes. Nevertheless, the presence of protein particles in the membrane patches deposited on amino-terminated flat surfaces and the integrity of the immobilized PSUs in terms of their spectral characteristics were confirmed by AFM and spectroscopic ellipsometry, respectively (Supporting Information). Moreover, the stability and functional activity of the immobilized photosynthetic proteins have already been addressed in previous studies.31-33 Likewise, membrane proteins such as G-protein(28) He, J. A.; Samuelson, L.; Li, L.; Kumar, J.; Tripathy, S. K. Langmuir 1998, 14, 1674-1679. (29) Choi, H. G.; Min, J. H.; Lee, W. H.; Choi, J. W. Colloids Surf., B 2002, 23, 327-337. (30) Chen, S. F.; Liu, L. Y.; Zhou, J.; Jiang, S. Y. Langmuir 2003, 19, 28592864. (31) Lee, I.; Lee, J. W.; Stubna, A.; Greenbaum, E. J. Phys. Chem. B 2000, 104, 2439-2443.

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coupled receptors have been immobilized on amino-coated glass slides to form protein arrays, and they were found to remain functionally active.34 Very recently, Ciobanu et al. demonstrated the selective deposition of Photosystem I on microcontact-printed SAM substrates,35 a related study that also explores the possibilities of constructing photosynthetic protein-based microdevices. Taken together, the above cited studies strongly suggest that DPN arrays can be used for functional studies of the PSUs and other membrane proteins. However, direct functional characterization of the LH1-RC complexes patterned on SAMs will be reported elsewhere. We would like to point out that the use of the DPN approach in designing amino-based chips for membrane protein analysis displays several important advantages. In the direct molecular writing process, a single contact of the scanning probe microscope (SPM) tip and the substrate (dot printing) is sufficient to generate surface features for selective protein immobilization, i.e., ultrahigh array densities can be achieved using parallel processing schemes (multitip cantilevers). The commercially available lithographic hardware and software also enables the design of complex nanopatterns using different inks for studying specific protein interactions, building “custom” protein assemblies, studying signal transduction, and so forth. For this purpose, more specific surface groups could be introduced via DPN using compounds with tailored molecular multivalency.36 Also, the same instrument could be used for the label-free readout, for example, by employing antibody-based recognition.7,8 Finally, recent studies on mica-supported PSUs15,27 demonstrated the power of AFM in resolving the structure of native membrane complexes. We believe that the micro- and nanopatterning of solid-supported membrane proteins by DPN can be combined with such structural and functional studies. For this reason, more detailed investigations are needed on the protein immobilization and orientation conditions, which will lead to the formation of oriented membrane protein assemblies on SAMs. Furthermore, the molecular templates for the membrane protein assemblies should preferably be generated on gold substrates that are smoother than those used in our study. Ultimately, it should be possible to address and manipulate individual membrane protein molecules in supporting nanopatterns with the SPM tip.

Conclusions We have demonstrated that HS(CH2)16NH2 can be used as ink for direct molecular writing. Regular microscopic and submicroscopic amino-terminated features were generated on gold under room conditions using DPN. The measured height of the molecular assemblies and their stability against post-filling with other thiol compounds was in agreement with the formation of a densely packed SAM. Furthermore, an enriched fraction separated from the cell membrane of Rhodobacter sphaeroides, which contains photosynthetic core complexes, was used as a model system for the selective recruitment to the array spots. This complex and crude membrane fraction was found to deposit selectively to the positively charged amino domains, whereas no membrane patches were observed in the PEG-terminated SAM regions. Although (32) Trammell, S. A.; Wang, L. Y.; Zullo, J. M.; Shashidhar, R.; Lebedev, N. Biosens. Bioelectron. 2004, 19, 1649-1655. (33) Das, R.; Kiley, P. J.; Segal, M.; Norville, J.; Yu, A. A.; Wang, L. Y.; Trammell, S. A.; Reddick, L. E.; Kumar, R.; Stellacci, F.; Lebedev, N.; Schnur, J.; Bruce, B. D.; Zhang, S. G.; Baldo, M. Nano Lett. 2004, 4, 1079-1083. (34) Fang, Y.; Frutos, A. G.; Lahiri, J. J. Am. Chem. Soc. 2002, 124, 23942395. (35) Ciobanu, M.; Kincaid, H. A.; Jennings, G. K.; Cliffel, D. E. Langmuir 2005, 21, 692-698. (36) Tinazli, A.; Tang, J. L.; Valiokas, R.; Picuric, S.; Lata, S.; Piehler, J.; Liedberg, B.; Tampe, R. Chemistry 2005, 11, 5249-5259.

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a number of technical and methodological issues have to be overcome before a more detailed topographic characterization can be developed on the single protein level, the present study demonstrates the convenience of DPN as a new tool for accurate and selective recruitment and manipulation of complex biomembrane assemblies.

support from the Graduate School Forum Scientium (SSF and Linko¨ping University) and a Marie Curie Fellowship of the European Community program IHP under Contract No. HPMTCT-2000-00130. The authors are also thankful to Jacob Piehler (Frankfurt University) for his help with the characterization of the membrane fraction and useful discussion.

Acknowledgment. We are thankful to Zˇ ivile˘ Katiliene˘ and Neal W. Woodbury (Arizona State University) for providing the chromatophore samples, and Johan Ekeroth for the aminothiol. This work was supported by the Swedish Foundation for Strategic Research (SSF) through the Biomimetic Materials Science program, the Swedish Research Council (VR), the Swedish Institute (SI) through the Visby program, and the Lithuanian State Science and Studies Foundation. Sˇ .V. acknowledges the

Supporting Information Available: Spectroscopic characterization of the photosynthetic core complexes from Rhodobacter sphaeroides in solution, contact mode AFM, and spectroscopic ellipsometry characterization of the membrane patches adsorbed on aminoterminated gold surfaces. This material is available free of charge via the Internet at http://pubs.acs.org. LA053367W