Morphology of Dry Solid-Supported Protein Monolayers Dependent on

The morphologies of dry MrgA protein monolayers on different solid ... MrgA is a dodecameric iron-storage protein which can form hexagonal, two-dimens...
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Langmuir 2006, 22, 7185-7191

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Morphology of Dry Solid-Supported Protein Monolayers Dependent on the Substrate and Protein Surface Properties Andreas Scho¨nafinger,† Anke Morbitzer,† Daniel Kress,† Lars-Oliver Essen,† Frank Noll,† and Norbert Hampp*,†,‡ Faculty of Chemistry and Materials Sciences Center, UniVersity of Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany ReceiVed NoVember 9, 2005. In Final Form: May 31, 2006 The morphologies of dry MrgA protein monolayers on different solid substrates prepared by a three-step procedure (adsorption from an incubation solution, rinsing to remove excess salt and protein, and drying) were investigated using atomic force microscopy. MrgA is a dodecameric iron-storage protein which can form hexagonal, two-dimensional (2D) crystalline monolayers on hydrophilic surfaces at low supersaturation. The formation of such two-dimensional crystals is heavily dependent on the pH and the salinity of the incubation solution as well as on the surface properties. Correlation of surface coverage with substrate charge, ionic strength, and pH indicates the dominance of electrostatic effects in adsorption, with the balance shifting between intermolecular repulsion and protein-substrate attraction. Close to the isoelectric point (pI) of MrgA, adsorption to the surface and the formation of 2D crystals are favored. By preparation of self-assembled monolayers of thiols with different end groups on template-stripped gold, the surface properties can be varied easily from high to very low protein affinity. The resulting patterns of the crystalline protein structures are novel and could be a starting point for further scientific study, e.g., solid-supported cocrystallization with DNA, and indeed developments with technological applications, such as mesostructured deposition of MrgAcaged nanoparticles.

Introduction The interaction of proteins with solid surfaces is a crucial phenomenon with implications for nanotechnology, biotechnology, and biomaterials science. Especially ordered twodimensional (2D) arrays of proteins are of considerable practical interest in fields as diverse as electron crystallography1 and the design of nanotechnological devices.2 2D protein crystals of adequate quality for the purpose of high-resolution structure analysis have been grown at the liquid-lipid interface.1 This has been achieved through the application of external laterally compressing forces (LB technique) or by spreading a protein solution at the liquid-air interface.3 Such 2D crystalline films can be transferred to solid substrates; however, this step is timeconsuming and often lacks reproducibility on large or structured surfaces. On the other hand, direct adsorption on solid substrates usually does not lead to large crystalline protein assemblies. Perfectly flat substrates, sufficient lateral mobility of individual proteins allowing nucleation on the substrate, and a natural propensity of the protein for two-dimensional self-assembly (e.g., S-layer proteins2) are regarded as being essential for 2D crystal growth. An understanding and control of the adsorption process and phenomena related to drying are necessary for efficient and reproducible preparation of dry protein layers. The amount of adsorbed protein is determined by the balance between proteinprotein and protein-surface interactions at the liquid-solid interface, which in turn is dependent on physicochemical properties of the protein, such as surface charge distribution or * To whom correspondence should be addressed. Phone: +49-6421282-5775. Fax: +49-6421-282-5798. E-mail: [email protected]. † Faculty of Chemistry. ‡ Materials Sciences Center. (1) Haas, H.; Brezesinski; G.; Mo¨hwald, H. Biophys. J. 1995, 68, 312-314. (2) Pum, D.; Neubauer, A.; Gyo¨rvary, E.; Sa´ra, M.; Sleytr, U. B. Nanotechnology 2000, 11, 100-107. (3) Yoshimura, H.; Scheybani, T.; Baumeister, W.; Nagayama, K. Langmuir 1994, 10, 3290-3295.

pI, the protein concentration, the pH and ionic strength of the incubating solution, and surface properties (e.g., charge, hydrophilicity). Models describing protein adsorption range from quiet simple equilibrium models, which include cluster formation,4 to more complicated dynamic simulations, which describe equilibrium and nonequilibrium kinetically frustrated states and the morphology of the adsorbate.5 However, none of these studies have comprehensively covered all aspects of protein adsorption.6 During drying of the adsorbate layer, capillary forces can induce lateral rearrangement of the proteins.7 Denkov8 introduced a qualitative model for the crystallization mechanism in drying colloidal films. The latter mechanism is driven by (a) the capillary attraction, which can be much larger than the thermal energy (kBT) even with nanometer-sized particles,9 and (b) the convective particle flux caused by the water evaporation from the preordered array. Consequently, investigation of the properties of dry adsorbates provides an essential objective for future work on capillary force-driven guided self-assembly of proteins on chemically patterned surfaces. In this study, we examine the influence of the pH and ionic strength of the incubation solution on the morphology and surface coverage of adsorbate monolayers of the dodecameric ferritinlike protein MrgA from Bacillus subtilis on surfaces with different properties with respect to charge and hydrophilicity. All parameters can be adjusted easily on different materials either by the use of different incubation solutions (e.g., on mica by adding bivalent cations) or by the preparation of self-assembled monolayers of surface-active reagents with different end groups (such as thiols on gold or silanes on silicon). Surface modification (4) Minton, A. P. Biophys. Chem. 2000, 86, 239-247. (5) Gray, J. J.; Bonnecaze, R. T. J. Chem. Phys. 2001, 114, 1366-1381. (6) Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110-115. (7) Kralchevsky, P. A.; Denkov, N. D. Curr. Opin. Colloid Interface Sci. 2001, 6, 383-401. (8) Denkov, N. D.; Velev, O. D.; Kralchevsky, P. A.; Ivanov, I. B.; Yoshimura, H.; Nagayama, K. Nature 1993, 361, 26. (9) Kralchevsky, P. A.; Paunov, V. N.; Ivanov, I. B.; Nagayama, K. J. Colloid Interface Sci. 1992, 151, 79-94.

10.1021/la0530182 CCC: $33.50 © 2006 American Chemical Society Published on Web 07/13/2006

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was followed by the adsorption of the protein from solution by rinsing, which basically removes not bound protein and excess salts from the incubation buffer, thus dramatically changing the physicochemical conditions during the drying step, e.g., largely decreasing the Debye screening length, and then slow drying. High-resolution AFM measurements revealed that the quaternary structure of the dodecameric MrgA does not collapse during adsorption and drying on the tested surfaces. It should be noted that MrgA is an unusually stable protein and that quite extreme conditions are required to observe dissociation. Even under SDS-PAGE conditions, MrgA does not fully dissociate. In our experiments, the stability of the MrgA dodecamers prevented denaturation upon adsorption. Owing to its rigidity and its nearly spherical shape, the adsorption step may be described as that of solid charged particles, rather than the more frequently studied protein adsorption systems which can denature. Indeed our results on surface coverage agree qualitatively with theoretical5,10 and experimental11 studies that deal with adsorption of proteins as spherical, charged particles. This indicates the dominance of electrostatic interactions in the adsorption step. However, at present no theoretical model comprehensively considers both the change of physicochemical conditions during rinsing on an adsorbate layer and cluster formation during drying. There are only a few examples in the literature where the quaternary structure is conserved upon adsorption. Adsorbed layers of horse spleen ferritin, which belongs to the same protein superfamily as MrgA but consists of 24 identical subunits instead of 12, has been studied in liquid, using liquid tapping mode atomic force microscopy (LTM-AFM).11 The radial distribution g(r) and surface coverage of wet ferritin films are in good qualitative agreement with those from random sequential adsorption (RSA) simulations for charged particles. In contrast to Johnson’s results, our dry protein layers appear to be more ordered but follow the same trends in surface coverage. Nygren12 found protein-clustered structures similar to ours, on hydrophobic methylized quartz surfaces, but did not correlate the resulting morphology to substrate properties. In our study, we concentrated on three different substrates: mica, gold, and silicon. All model substrates are easily prepared without contamination and with atomically flat surfaces. Gold is in the form of ultraflat template-stripped gold (TSG).13 Furthermore, gold surfaces can be modified without much effort by chemisorption of alkanethiols. In the case of silicon, chemical modification can as well be easily performed through the use of silanes. Experimental Section Protein. MrgA from B. subtilis 16814 (PDB ID 2CHP) is a dodecameric, ferritin-like, iron-storage protein capable of binding DNA. As known from the three-dimensional (3D) crystal structure of the dodecameric MrgA complex, it forms a hollow sphere with an acidic interior, and basic lysine residues (at the N-terminus) are located on the outer surface.15 MrgA crystallizes in space group P213, and the crystal lattice is built up by stacked hexagonal layers of the protein complex. At pH 7 the calculated net charge of the complex amounts to -168e. Figure 1 shows the calculated surface (10) Oberholzer, M. R.; Wagner, N. J.; Lenhoff, A. M. J. Chem. Phys. 1997, 107, 9157-9167. (11) Johnson, C. A.; Yuan, Y.; Lenhoff, A. M. J. Colloid Interface Sci. 2000, 223, 261-272. (12) Nygren, H.; Stenberg, M. Biophys. Chem. 1990, 38, 67-75. (13) Wagner, P.; Hegner, M.; Gu¨ntherodt, H.-J.; Semenza, G. Langmuir 1995, 11, 3867-3875. (14) Chen, L.; Keramati, L.; Helmann, J. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 8190-8194. (15) Grant, R. A.; Filman, D. J.; Finkel, S. E.; Kolter, R.; Hogle, J. M. Nat. Struct. Biol. 1998, 5, 294-303.

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Figure 1. Calculated surface potential of the dodecameric MrgA complex: (left) cutaway view showing the inner surface of the hollowsphere-like protein dodecamer; (right) outer surface; (red) negative charge (-75.3 kJ/e); (blue) positive charge (+75.3 kJ/e). potential of MrgA at pH 7 and a (monovalent) salt concentration of 100 mM. The calculation was performed with the program GRASP.16 Iron is stored in the protein complex’s interior as ferrihydrite. MrgA belongs to the group of Dps proteins (DNA-protecting proteins from starved cells).17 Many Dps proteins bind DNA in an unspecific manner with respect to the DNA nucleic acid sequence and show a high affinity for DNA.18,19 The formation of Dps-DNA cocrystals in vitro and in vivo is well-known.20,21 The precise mode of the DpsDNA interaction has yet to be fully elucidated. Only the dependence of the Dps-DNA interaction on the salt concentration has been observed in the cell, where Dps and DNA form a stable complex protecting the DNA double strand from mutagenic chemicals.20,21 Frenkiel-Krispin et al.20 proposed that Dps dodecamers cannot bind DNA molecules directly and that complex formation is mediated through ion bridges formed by Mg2+. Other studies revealed, however, that the highly flexible lysine-rich N-terminus plays a major role in Dps-DNA interaction.15 The fact that members of the Dps family not possessing a positively charged N-terminus fail to bind DNA in agarose gel mobility assays supports this finding.22-25 Dps selfaggregation strongly reflects the properties of the N-terminus. Escherichia coli Dps requires at least 150 mM NaCl to remain in solution at pH values around 7, whereas Dps proteins lacking a positively charged N-terminus are often fully soluble even in the absence of salt.25 In solution, the identical subunits of MrgA form the rigid dodecameric MrgA. In contrast to proteins with a high tendency to self-assemble in 2D arrays such as S-layer proteins, MrgA only shows a low tendency to do so due to the lack of strong proteinprotein interactions. MrgA forms hexagonal monolayers on some hydrophilic substrates, but because of the weak protein-protein interactions, the physicochemical properties of the physisorption process have to be controlled more carefully. This can be accomplished by varying the composition of the incubation solution as well as the surface properties of the substrates by, e.g., selfassembled monolayers (SAMs) on gold comprising alkanethiols with different end groups. A covalent binding via surface-accessible residues to a reactive surface was not performed because it often lacks regiospecifity, and (16) Nicholls, A. GRASP: Graphical representation and analysis of surface properties; Columbia University: New York, 1992. (17) Chen, L.; Helmann, J. D. Mol. Microbiol. 1995, 18, 295-300. (18) Martinez, A.; Kolter, R. J. Bacteriol. 1997, 179, 5188-5194. (19) Sen, A.; Dwivedi, K.; Rice, K. A.; Bullerjahn, G. S. Arch. Microbiol. 2000, 173, 352-357. (20) Frenkiel-Krispin, D.; Levin-Zaidman, S.; Shimoni, E.; Wolf, S. G.; Wachtel, E. J.; Arad, T.; Finkel, S. E.; Kolter, R.; Minsky, A. EMBO J. 2001, 20, 11841191. (21) Minsky, A.; Shimoni, E.; Frenkiel-Krispin, D. Nat. ReV. Mol. Cell Biol. 2002, 3, 50-60. (22) Bozzi, M.; Mignogna, G.; Stefanini, S.; Barra, D.; Longhi, C.; Valenti, P., Chiancone, E. J. Biol. Chem. 1997, 272, 3259-3265. (23) Papinutto, E.; Dundon, W. G.; Pitulis, N.; Battistutta, R.; Montecucco, C.; Zanotti, G. J. Biol. Chem. 2002, 277, 15093-15098. (24) Zanotti, G.; Papinutto, E.; Dundon, W. G.; Battistutta, R.; Seveso, M.; Del Giudice, G.; Rappuoli, R.; Montecucco, C. J. Mol. Biol. 2002, 323, 125-130. (25) Ceci, P.; Cellai, S.; Falvo, E.; Rivetti, C.; Rossi, G. L.; Chiancone, E. Nucleic Acids Res. 2004, 32, 5935-5944.

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as a consequence, a distinct orientation and order of the immobilized protein cannot be achieved. In contrast, weak electrostatic interactions allow lateral diffusion of the physisorbed protein on the surface, so the protein can self-assemble in two dimensions to supramolecularly ordered structures. Chemicals. HPLC-grade solvents were purchased from Fisher Scientific (Leicestershire, U.K.) and used without further purification. Thiols with various terminating functional groups such as 11mercapto-1-undecanol (97%, Aldrich, Taufkirchen, Germany), 1H,1H,2H,2H-perflourodecanethiol (99%, Fluorochem, Derbyshire, U.K.), and 1-hexadecanethiol (>95%, Fluka, Buchs, Switzerland) were used as provided by the manufacturer. 16-Mercaptohexadecanoic acid (90%, Aldrich) was purified by column chromatography on silica gel and eluted with dichloromethane/acetone (1:1, v/v, Rf ) 0.66). The substance was further purified by recrystallization from ethanol. 16-Mercaptohexadecanoic acid 2-[2-(2-hydroxyethoxy)ethoxy]ethyl ester was synthesized as described by Tinazli et al.26 Solutions of 1 mM thiols were prepared in argon-saturated absolute ethanol. Aliquots were stored at -20 °C. Sample Preparation. Epoxy-glue-supported TSG substrates were prepared according to the procedure published by Wagner et al.13 Onto mica muscovite (Plano GmbH, Wetzlar, Germany), freshly cleaved along the (001) plane, was slowly vapor-deposited an approximately 200 nm thick gold layer (99.99%, Degussa, Hanau, Germany) at a pressure of less than 10-4 Pa. The fragile gold layer was reinforced by gluing a float glass slide onto it using an epoxy glue (epo-tec 377, Polytec, Waldbronn, Germany). The glue was cured by heating to 150 °C for 2 h. The resulting glass-epoxy glue-Au-mica sandwiches were stored at room temperature without any detectable loss of quality. As a final step, the TSG surfaces were cleaved mechanically immediately before use. By this process polycrystalline Au(111) surfaces were obtained with a surface roughness Rrms of less than 0.6 nm as measured by AFM. Muscovite mica surfaces used for protein adsorption were prepared by cleaving the substrate immediately prior to incubation. Silicon wafer surfaces were cleaned using piranha solution (96% H2SO4: 30% H2O2 ) 2:1) followed by thorough rinsing with distilled water. Amination of cleaned silicon surfaces was performed by incubation of the surface in a 2% solution of (3-aminopropyl)triethoxysilane (APTES) in dichloromethane for 1 h, followed by thorough rinsing with doubly distilled water. Self-assembled monolayers of thiols on TSG were prepared immediately prior to protein adsorption by incubation of the gold surface in a 0.1 mM ethanolic solution of the thiol for 3 h followed by washing with ethanol and doubly distilled water. Incubation with protein solution (1-100 ng/µL, pH ranging from 3.5 to 7.5 in different buffer systems) was allowed for 1 h. All samples were washed with distilled water and allowed to dry. The amount of protein that is theoretically necessary to completely cover a given surface with MrgA is approximately 3.5 ng/mm2. All preparations were performed at protein concentrations above this theoretical value. MrgA was heterologously expressed in E. coli using autoinduction at 30 °C for 24 h. The cells were harvested and resuspended in buffer solution at pH 7.5 (25 mM HEPES, 25 mM NaCl). After lysis of the cells the supernatant was heat denatured (85 °C for 10 min). The dodecameric protein MrgA remained soluble, and most of the E. coli proteins were removed by centrifugation (5 min, 14000g). The supernatant, containing reasonably pure MrgA (∼80% homogeneity), was applied to a DEAE-Sepharose anion exchange matrix. The eluted material was concentrated to 40 mg/mL using a Vivaspin 6 concentrator (Vivascience, Hannover, Germany, MWCO ) 10000). As a final purification step, a prepacked Superdex S-200 HR column was used. The most prominent peak, which corresponds to the dodecameric MrgA complex, was collected. The buffer was exchanged with 10 mM HEPES (pH 7.5) and the sample concentrated again to a final concentration of 22 mg/mL. The protein concentration was measured using a modified Bradford test.

AFM Imaging. AFM images have been collected using a Digital Instruments Multimode atomic force microscope (Veeco Instruments, Santa Barbara, CA) equipped with a Nanoscope IV controller and an E-scanner. Imaging was performed in tapping mode in air immediately after sample preparation. Single-crystalline silicon tips were used (RTESP, NanoDevices, Veeco Instruments). All images presented are raw data or have only undergone minimal manipulation of first- or second-order two-dimensional flattening. Molecular resolution was achieved by successive reduction of the drive amplitude and manual return into feedback. Details of the sample preparation are given in the figure captions.

(26) Tinazli, A.; Tang, J.; Valiokas, R.; Picuric, S.; Lata, S.; Piehler, J.; Liedberg, B.; Tampe´, R. Chem.sEur. J. 2005, 11, 5249-5259.

(27) Ren, B.; Tibbelin, G.; Kajino, T.; Asami, O.; Ladenstein, R. J. Mol. Biol. 2003, 329, 467-477.

Results and Discussion MrgA monolayers were prepared on a variety of substrates with the aim of examining the protein binding characteristics as affected by different surface properties such as hydrophilicity and surface charge. The pH as well as the ionic strength of the incubation solution was also varied, with a view to examine the influence of the protein net charge on its surface binding tendency. The first model substrate chosen was muscovite mica. Mica offers a highly hydrophilic, negatively charged surface. Figure 2 shows two typical AFM images of crystalline MrgA monolayers (Figure 2a,c). The two-dimensional fast Fourier transformation (FFT) pattern corresponding to Figure 2a is shown in Figure 2b. The hexagonal lattice formed by MrgA can be identified easily (Figure 2b,c). The line scan in Figure 2a shows that the monolayer has a thickness of approximately 9 nm, a value which is in good agreement with the dimension of MrgA from crystal data (9.04 nm). FFT analysis (Figure 2b) of the whole image does not show sharp spots (Figure 2a) but nevertheless demonstrates that the crystal orientation remains fairly constant over the whole 1 µm2 area, even though the crystalline area has some gaps. In the hexagonal two-dimensional lattices an average protein-protein distance of about 9 nm can be measured from the FFT analysis, which corresponds well to the layerlike packing of Dps molecules in the unit cell of three-dimensional crystals derived from Dpslike ferritin X-ray crystallographic data.15,27 It is likely that the packing on the surface is similar to that in the crystal and that the same interactions between dodecamers control both processes. Figure 2c shows an AFM image of a second sample taken at a higher magnification. Here the hexagonal package can be identified clearly. Furthermore, it has been found that MrgA forms hexagonally packed protein monolayers under different adsorption conditions (pH ranging from 3.5 to 8.0, buffer salt concentrations from 10 to 100 mM), possibly due to the surface properties of mica. Desorption of MrgA from mica during extended rinsing was observed in the absence of Mg2+. Reproducibility on mica sample preparation is dependent on the method of rinsing and drying. On slowly dried (approximately within 0.5 h) mica different morphologies and surface coverages were observed on the same sample. Besides large crystalline and clustered morphologies similar to those on the MHDA samples shown in Figure 6, regions without any protein coverage could be found. We assume a desorption-adsorption mechanism and/or lateral forces at the liquid-air interface during the drying process may be responsible for this heterogeneous distribution of protein on the surface. Samples blow-dried after rinsing had a more homogeneous surface coverage but still showed the characteristic protein clusters. At the conditions found to be optimal for mica as a substrate in terms of surface coverage MrgA shows no tendency to form crystalline monolayers on gold. This result corresponds to findings

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Figure 3. Chemical structures of the thiols used for SAM preparation on TSG. MHDA ) 16-mercaptohexadecanoic acid, MUDOH ) 11-mercaptoundecanoic acid, HDT ) 10-mercaptohexadecane, PFDT ) 1H,1H,2H,2H-1-mercaptoperfluorodecane, and MHDAEG3 ) 16-mercaptohexadecanoic acid 2-[2-(2-hydroxyethoxy)ethoxy]ethyl ester. Table 1. Coverage of Different Surfaces with MrgA after Incubation from Solution with and without Mg2+ Cations at Two Different pH Values and a Protein Concentration of 1 ng/µLa substrate surface

pH 5.0

pH 5.0, 20 mM MgCl2

pH 7.5

pH 7.5, 20 mM MgCl2

MHDA PFDT MUDOH HDT MHDA-EG3

++, a ++, a ++, c +, a --

+, c ++, a +, c +, a --

-, c +, a -, s o, a --

+, c ++, a o, c +, a --

a Coverage: ++, well-covered; +, covered; o, slightly covered; -, poorly covered; - -, uncovered. Morphology: c, clustered; s, sparse; a, amorphous. The buffer was 10 mM Tris.

for another iron storage protein, ferritin.28 However, modification of gold surfaces is accomplished through the use of thiols, which chemisorb rapidly on gold, thus developing and forming SAMs. Preparation of SAMs from thiols is a common and frequently used procedure of modifying the surface properties of gold.29-35 To optimize the formation of hexagonally arranged MrgA monolayers on gold, we varied both the incubation solution (pH, ionic strength, MrgA concentration) and the surface properties, which were tuned by different end groups of the thiols (carboxy, hydroxy, alkane, perflouroalkane, and oligo(ethylene glycol)) used for SAM preparation (see Figure 3 for the chemical structures of the thiols used). For this screening of adsorption conditions low concentrations of the protein were chosen, leading to smaller protein coverage of the surfaces. This facilitates analysis of the effects of pH and ionic strength on the morphology and surface coverage, in comparison to that of more densely covered samples. Table 1 summarizes the experiments performed on five different surfaces Figure 2. AFM images (a, c) of an MrgA monolayer on mica muscovite and FFT analysis (b) of the image shown in (a). The substrate was fully covered with a 10 ng/µL solution of MrgA in 10 mM Tris buffer (pH 5.0). The hexagonal lattice can be identified easily in (c). The height profile of a line scan (a) marked by the white line shows the height of an MrgA monolayer. FFT analysis (b) calculated for the full image (a) shows that the crystalline structure throughout the whole 1 µm2 area has nearly the same lateral orientation, despite some gaps in the crystalline area. From the FFT analysis an average protein-protein distance of approximately 9 nm has been determined.

(28) Caruso, F.; Furlong, D. N.; Kingshott, P. J. Colloid Interface Sci. 1997, 186, 129-140. (29) Nuzzo, R. G.; Zegarski, B. R.; Dubois, L. H. J. Am. Chem. Soc. 1987, 109, 733. (30) Strong, L.; Whitesides, G. M. Langmuir 1988, 4, 546-558. (31) Bain, C. D.; Whitesides, G. M. Science (Washington, D.C.) 1988, 240, 62-63. (32) Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335. (33) Ulman, A.; Eilers, J. E.; Tillman, N. Langmuir 1989, 5, 1147-1152. (34) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87-96. (35) Ulman, A. Chem. ReV. 1996, 96, 1533-1554.

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Figure 4. AFM images of two SAMs on TSG incubated with MrgA solution (pH 5.0, 1 ng/mL, 10 mM Tris): (a) MHDA SAM, dense but amorphous protein coverage of the surface; (b) MHDA-EG3 SAM, no proteins adsorbed to the surface.

with four different incubation solutions at a constant protein concentration of 1 ng/µL. Figure 4 shows two selected AFM images of different SAMs used for protein adsorption at pH 5.0 after incubation. On MHDA a large amount of protein adsorbs (Figure 4a), whereas MHDA-EG3 fully protects the surface from protein deposition (Figure 4b). Two general tendencies were revealed: (i) Sample surfaces prepared at a pH close to the pI (4.6) of MrgA display better protein coverage than samples prepared at pH 7.5 and (ii) a higher ionic strength at pH close to the pI leads to less protein coverage, whereas at a high pH (7.5) denser surface coverage is observed. At pH close to the pI the total charge of MrgA is nearly zero, whereas at pH 7.5 MrgA is strongly negatively charged. Negative charges are predominantly located at the inner surface and positive charges on the lysine-rich outer surface of the hollow-sphereshaped dodecameric MrgA complex. For uncharged particles, electrostatic double-layer repulsion is minimized; thus, we observed the highest surface coverage near the pI, when proteinsurface interaction is attractive and protein-protein repulsion is

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low. At a short protein-protein distance, hydrophobic interaction stabilizes tightly packed protein clusters. The repulsive electrostatic interaction between the proteins themselves, and the protein and the surfaces with negative surface charge, explains the decreasing amount of adsorbate at pH 7.5 and at low salt concentrations. The surface charge of MHDA is supposed to vary with pH. It is known from the literature36,37 that the pKa value of surface-bound carboxylic acid groups increases significantly compared to the value in solution. Thus, at pH 5.0 the MHDA surface is almost uncharged, whereas it is negatively charged at pH 7.5. This explains the differences in the amount of adsorbed MrgA at low salt concentrations at the two different pH values. The surface of MUDOH and that of PFDT are also negatively charged and follow the same trends in surface coverage as mica or MHDA at pH 7.5. For adsorbing conditions close to the isoelectric point the positively charged outer shell of the protein complex electrostatically binds directly to oppositely charged surfaces, such as mica, MHDA, PDFT, and MUDOH. In this case, a higher ionic strength obstructs the approach of the protein to the surface due to the formation of a repelling electrostatic double layer. In contrast, the adsorption of MrgA at pH 7.5 is promoted by higher salt concentrations. The participation of Mg2+ cations in the adsorption process overcomes the repulsive forces between the protein and the surface by charge screening. Interestingly, it is this mode of cation-bridged interaction that was earlier postulated as being a major factor in Dps-DNA interaction at the almost neutral pH conditions of the crystal.20 Under low salt conditions, the net charge in the contact zone between the protein and the surface (since that region has a relatively low dielectric permittivity) leads to a high electrostatic potential and is therefore highly unfavorable.38 On hydrophobic substrates such as HDT, at pH 5.0, amorphous, rodlike agglomerates were obtained. The volume of these rods equals approximately the volume of a 30% dense protein monolayer. We therefore assume that the wet and viscous monolayer contracts and partially slides off the sample during drying. In contrast, at pH 7.5 MrgA covers the surface to some extent, forming an amorphous monolayer. The difference in the adsorption behavior, as a function of pH, may be explained by electrostatic protein-protein interactions. It is well-known39 that surface-bound poly(ethylene glycol)s prevent protein adsorption. Therefore, it was not surprising that MHDA-EG3 proved to be an MrgA-resistant surface in a wide range of physicochemical conditions. At present the reason poly(ethylene glycol) end groups impede protein adsorption to the surface is still under debate.40 As indicated by our experiments, pH and charge are factors that affect surface coverage. Further experiments on moderately hydrophilic silicon oxide show that resistance against protein adsorption can be obtained not only through changing the physicochemical conditions or using ethylene glycols but also by changing the surface charge by amination. Silicon surfaces were used as model substrates because the oxide layer of wafer surfaces can be modified easily by amine-terminated silanes. Figure 5a shows an MrgA monolayer deposited on the silicon oxide layer of a Si(100) wafer. The surface is well covered with (36) Hu, K.; Bard, A. J. Langmuir 1997, 13, 5114-5119. (37) Konek, C. T.; Musorrafiti, M. J.; Al-Abadleh, H. A.; Bertin, P. A.; Nguyen, S. T.; Geiger, F. J. Am. Chem. Soc. 2004, 126, 11754-11755. (38) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1979, 71, 350-366. (39) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 1071410721. (40) Siegers, C.; Biesalski, M.; Haag, R. Chem.sEur. J. 2004, 10, 28312838.

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Figure 6. Series of AFM images of different MrgA preparations on SAMs of MHDA. The coverage and structure of the MrgA layers change depending on the pH of the incubation solution: (a) pH 3.5; (b) pH 3.75; (c) pH 4.0; (d) pH 5.0. Incubation conditions: 10 ng/mL MrgA in 0.1 M NH4H2PO4. Image sizes are 1 µm2.

Figure 5. AFM images of two silicon surfaces incubated with MrgA (pH 4.0, 10 ng/ mL, 0.1 M NH4H2PO4): (a) cleaned silicon; protein clusters cover the surface; (b) silicon surface modified with APTES; no protein adsorbs.

the protein. Under identical conditions but when using an APTEStreated silicon oxide surface, no protein adsorption is observed (Figure 5b) because the surface is now strongly positively charged. The latter clearly demonstrates that protein adsorption can be regulated by the substrate charge. A second set of experiments was performed to obtain denser MrgA monolayers with large monocrystalline areas. In the previous experiments, MHDA on gold proved to be the most promising SAM-modified substrate with respect to MrgA cluster formation and surface coverage. A further reason for focusing on modified TSG surfaces in general is that it may be easily modified in a structured way through microcontact printing (µCP).41 Since below the pI of MrgA the protein is positively charged, experiments were performed near the pI and below. As shown in Figure 6 the surface becomes more and more densely covered with protein with increasing pH (for an MrgA concentration of 10 ng/µL). Close to the isoelectric point of the protein, larger (41) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498-1511.

Figure 7. AFM image of an MHDA SAM on TSG incubated with MrgA solution (pH 4.0, 100 ng/mL, 0.1 M NH4H2PO4). The surface is well covered, but agglomeration also occurs.

vertical aggregates can be found on the surface (pH 5.0). On some mica samples, and under these conditions, the protein formed two hexagonal layers packed vertically in 0.2 µm spots. However, on the MHDA surfaces, amorphous vertical aggregation was observed. At a pH of 4.0 the best coverage was achieved without vertical aggregation; therefore, the effect of the MrgA concentration on the cluster formation was further investigated. Obviously, more densely packed crystalline arrays were observed at higher MrgA concentrations. At significantly higher MrgA concentrations (100 ng/µL), colloidal agglomeration of the protein in the solution as well as on the surface (Figure 7) can be observed. Different washing and drying conditions do not affect the MrgA layers on MHDA SAMs, and desorption is not observed. The morphology and surface coverage were reproducible on these substrates. In contrast to mica, which is regarded as an almost defect free, atomically flat substrate, SAM-modified TSG is a soft surface and exposes some defects, resulting in a larger contact area

Morphology of Solid-Supported Protein Monolayers

between the protein and the substrate. In the latter, some proteins are probably tethered to the substrate, while others may diffuse laterally. Although in general mica was more densely covered with MrgA, indicating a stronger protein-surface interaction in the adsorption step, desorption becomes possible to a greater extent upon removal of ions during rinsing. In contrast, no desorption from MHDA templates was observed. Interestingly, the crystal lattice orientation is sustained across grain boundaries on mica samples as demonstrated by FFT (Figure 2b). The MHDA samples showed smaller, connected MrgA clusters which did not have a preferential large-scale orientation. Owing to the protein’s lower lateral mobility and defects of the substrate, this is just what we expected. It is likely that the protein packing on the dried surfaces in both cases is similar to that in the 3D crystal and that they exploit the same interactions between dodecamers. Although our results on surface coverage follow the same trends as published in an LTM-AFM study by Lenhoff,11 striking differences in morphology are obvious. Whereas the LTM-AFM images display a sparse distribution of the protein, our mica, MHDA, and silicon samples display a clustered or even crystalline morphology. Further experiments have to be performed to distinguish whether the removal of excess salts induces surface crystallization in the rinsing step or crystallization is simply capillary force driven.

Summary and Conclusions The adsorption of MrgA on different substrates under a variety of physicochemical conditions has been examined. The crystallization of MrgA to a monolayer depends strongly on the physicochemical conditions, in particular on the pH value and ionic strength of the buffer used during physisorption and on the hydrophilicity and the surface charge of the substrate. The surface charge of the protein and the substrate can be controlled easily, resulting in optimal protein adsorption conditions or in total protein repulsion from the substrate surface. Table 2 summarizes the main findings. From the findings presented here we conclude that surface charge distribution of the substrates and the protein is one of the main factors that influence surface coverage and the formation of a monolayer. Maximum MrgA affinity for the surfaces was

Langmuir, Vol. 22, No. 17, 2006 7191 Table 2. Optimized Coverage and Morphology of Different Surfaces with MrgA Achieved after Incubation from Solutiona substrate surface mica silicon wafer TSG

a

optimized MrgA coverage freshly cleaved ++, crystalline 10 mM Tris, pH 5 cleaned with piranha solution ++, clustered pH 4.0, 0.1 M NH4H2PO4 MHDA-treated ++, clustered pH 4.0, 0.1 M NH4H2PO4

MrgA adsorption resistance none APTES-modified - -, sparse pH 4.0, 0.1 M NH4H2PO4 MHDA-EG3-modified -various conditions

Coverage: ++, well-covered; - -, uncovered.

found under conditions where the charge of the protein just compensates for the substrate surface charge, and no additional bivalent cations are needed to mediate the binding between the protein and surface. This is in accordance with models for other proteins presented in the literature.38 At the time being it is not possible to monitor the rearrangement of adsorbed proteins during drying. However, high-resolution measurements in a liquid11 environment and on dried samples12 indicate that capillary forces alter the surface morphology. In this study on dried MrgA adsorbates, we found that the formation of crystalline, clustered, amorphous, and sparse protein assemblies is dependent on the substrate properties and incubation conditions. SAMs on TSG are promising candidates for preparing mesoand nanoscopic structured surfaces with areas well suited for protein adsorption (e.g., MHDA) and areas with no affinity for proteins (e.g., MHDA-EG3). It is expected that, by using structured SAMs on TSG, it will become possible in the future to simultaneously perform and analyze crystallization in thousands of microarrays and to study nanopatterning processes for the preparation of layered nanostructures. Acknowledgment. We thank A. Tinazli for providing various thiols (i.e., MHDA-EG3). This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through Grant HA 2906/4-2. LA0530182