NANO LETTERS
Recognition Imaging and Highly Ordered Molecular Templating of Bacterial S-Layer Nanoarrays Containing Affinity-Tags
2008 Vol. 8, No. 12 4312-4319
Jilin Tang,† Andreas Ebner,† Helga Badelt-Lichtblau,‡ Christine Vo¨llenkle,‡ Christian Rankl,† Bernhard Kraxberger,† Michael Leitner,† Linda Wildling,† Hermann J. Gruber,† Uwe B. Sleytr,‡ Nicola Ilk,‡ and Peter Hinterdorfer*,† Institute of Biophysics, Johannes Kepler UniVersity of Linz, 4040 Linz, Austria, and Center for NanoBiotechnology, UniVersity of Natural Resources and Applied Life Sciences, 1180 Vienna, Austria Received July 15, 2008; Revised Manuscript Received September 16, 2008
ABSTRACT Functional nanoarrays were fabricated using the chimeric bacterial cell surface layer (S-layer) protein rSbpA fused with the affinity tag StreptagII and characterized using various atomic force microscopy (AFM) techniques in aqueous environment. The accessibility of Strep-tagII was verified by single-molecule force spectroscopy studies employing Strep-Tactin as specific ligand. Simultaneous topography and recognition imaging (TREC) of the nanoarray yielded high resolution maps of the Strep-tagII binding sites with a positional accuracy of 1.5 nm. The nanoarrays were used as template for constructing highly ordered molecular binding blocks.
Crystalline bacterial cell surface layers (S-layers) represent a unique self-assembly system optimized during billions of years of biological evolution.1-4 The intrinsic ability of S-layers to self-assemble allows for in vitro formation of isoporous two-dimensional (2D) protein lattices in suspension, on lipid film, on liposome, and on solid supports.1,5 Molecular self-assembly systems that exploit the molecularscale manufacturing precision of biological systems are prime candidates for controlled “bottom-up” production of defined nanostructures.6-8 Since one of the most relevant areas in nanobiotechnology concerns technological utilization of selfassembly systems, it appeared useful to advance S-layer technology by the construction of recombinant functional S-layer fusion proteins. As shown before,9-15 fusion of functional domains to S-layer proteins did not interfere with self-assembly and recrystallization properties of the S-layer protein moiety. The aim of the construction of S-layer fusion proteins was to combine the self-assembly properties of S-layer proteins with a broad spectrum of specific functions (e.g., ligands, antibodies, antigens, enzymes) providing an unsurpassed precision in spatial control and of alignment of functions encoded in proteins.1,10,11,13 * To whom correspondence should be addressed. E-mail: peter.
[email protected]. † Johannes Kepler University of Linz. ‡ University of Natural Resources and Applied Life Sciences. 10.1021/nl802092c CCC: $40.75 Published on Web 11/05/2008
2008 American Chemical Society
While high-resolution imaging of 2D protein nanoarrays is well established, the accurate location of single functional sites within the nanoarray lattice represents a new challenge. Because of the pN force sensitivity and nanometer positional accuracy, atomic force microscopy (AFM) has emerged as a powerful tool for imaging the surface of biological samples16,17 and exploring the forces between receptors and ligands at the single molecule level on isolated molecules as well as on cellular surfaces under physiological conditions.18 The recently developed technique “Topography and RECognition imaging” (TREC) allows for simultaneous mapping topography and single recognition sites of specific molecules present on complex biological sample surfaces with nanometer-scale precision.19,20 Compared to techniques such as force-volume mapping,21,22 TREC is faster and offers significantly better lateral resolution.18 In TREC experiments, the AFM tip is upgraded to a monomolecular sensor. For this purpose, a specific ligand is tethered to the tip via a flexible polyethylene glycol (PEG) chain.23,24 Molecular recognition signals are detected during dynamic force microscopy imaging.25 While laterally scanning over the biological sample containing corresponding receptor sites, a reduction in the oscillation amplitude of the modified AFM tip occurs while the ligand and the receptor are bound to each other. In this way, a topographical image and a map of recognition sites are simultaneously obtained from a single
scan. Moreover, recognition site mapping is efficient, reproducible, and specific, as shown by preceding investigations of receptor-ligand interaction,20 chromatin remodeling,25 and receptor site location on cells.26 In the present study, Strep-tagII, an eight amino acid peptide and artificial ligand for Strep-Tactin (a genetically engineered streptavidin variant with higher affinity toward Strep-tagII compared to streptavidin27) was fused to a C-terminally truncated form of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177. SbpA is one of the most extensively studied S-layer proteins, with the ability to self-assemble into a square (p4) lattice.1,5,12,28 In previous studies it was found that the fused Strep-tagII did not interfere with the self-assembly properties of S-layer fusion protein rSbpA-Strep-tagII and that the Strep-tagII was located on the exposed surface of the S-layer lattice.15 So far, mapping the functional groups in a nanoarray formed by rSbpA-StreptagII with nanometer resolution has not been reported. Here, we used TREC to investigate fusion protein rSbpA-StreptagII nanoarrays, so as to localize the functional Strep-tagII group in the recognition images with nanometer scale. We also developed a method to construct controlled protein blocks using an rSbpA-Strep-tagII crystalline lattice as a biotemplate. Single-molecule force spectroscopy was first applied to investigate the interaction strength between Strep-Tactin and Strep-tagII on the rSbpA-Strep-tagII lattice. Before starting single-molecule force spectroscopy experiments, rSbpAStrep-tagII was verified to self-assemble into monolayer protein 2D nanoarrays with square lattice symmetry on the silicon chip by AFM MAC mode (Agilent Technologies, Chandler, USA) imaging (Figure 1A). Then, an AFM tip was covalently functionalized with Strep-Tactin via a short, elastic linker (PEG, ∼2 nm, see Figure 1B). Figure 1C shows a typical force distance cycle containing a recognition event between Strep-Tactin and Strep-tagII. The functionalized AFM tip was brought near to the rSbpA-Strep-tagII monolayer (Figure 1C, dotted). A possible formation of a StrepTactin-Strep-tagII complex resulted in a stretching of the crosslinker during withdrawing the AFM tip from the surface (Figure 1C, solid). The force increased until a particular unbinding force was reached at which the complex disrupted and the tip returned to its zero position. Force distance cycles (n ) 1000) were collected by repeatedly approaching and subsequently retracting the functionalized AFM tip from an rSbpA-Strep-tagII monolayer surface and measuring the unbinding force in each cycle. An experimental probability density function (pdf) of the unbinding forces was constructed29 from 170 single unbinding force values which were acquired from the force distance curves containing an unbinding event. The most probable unbinding force (the maximum of the distribution) between Strep-tagII and StrepTactin was about 37 pN at a loading rate of 337 pN s-1, with a binding probability (probability of finding an unbinding event in force-distance cycles) of about 17% (Figure 1D, solid line). When free Strep-tag II peptides (36 µg/ml) were present in solution, the recognition events in the force distance cycles disappeared (Figure 1C inset). This led to a Nano Lett., Vol. 8, No. 12, 2008
decrease of the binding probability to about 6% (Figure 1D, dotted line), proving the specificity of the interaction between Strep-tagII on the lattice of rSbpA-Strep-tagII and StrepTactin. For lateral imaging of the recognition sites on the nanoarray, we applied a recently developed technique, TREC,20,25 to identify the location of Strep-tagII in the square S-layer lattice with nanoscale resolution. The topography images of the nanoarrays with cocrystallized S-layer proteins rSbpA-Strep-tagII and wild-type SbpA were acquired with dynamic mode AFM, whereby an alternating magnetic field drove the oscillation of magnetically coated cantilever. The 2D nanoarrays of cocrystallized S-layer proteins showing a square lattice symmetry with a center to center spacing of ∼14 nm, which is typical for the S-layer protein SbpA,4,15was observed in the topography images (Figure 2A). Figure 2B shows the simultaneously acquired recognition image, generated by scanning with a Strep-Tactin coupled AFM tip over the surface of the cocrystallized rSbpA-Strep-tagII/wild-type SbpA lattice. In this experiment, the PEG tether gives StrepTactin the possibility to freely move in a defined space and to reorient in order to properly bind to Strep-tagII on the S-layer lattices. When Strep-Tactin on the tip recognized Strep-tagII, the oscillating tip response was altered. This change was converted into the recognition signal20,25 which displayed the location of Strep-tagII in the S-layer nanoarrays. The dark spots in the recognition image (Figure 2B) indicate where recognition events between Strep-Tactin and Strep-tagII occurred. They obviously reflect the distribution of the fusion protein rSbpA-Strep-tagII in the cocrystallized S-layer lattice consisting of rSbpA-Strep-tagII and wild-type SbpA (at a molar ratio of 1:7). Although located all over the nanoarray surface of the cocrystallized S-layer proteins, the dark spots were randomly scattered, indicating that StrepTactin recognized only S-layer proteins with Strep-tagII in the cocrystallized S-layer lattice. The specificity of the recognition signals was proven by acquiring topography and recognition images at the same place after blocking StrepTactin on the tip (Figure 2C and D). The dark spots in the recognition image were completely abolished after injection of free Strep-tagII into the AFM fluid cell during imaging (Figure 2D). In this case, the ability of the tip-tethered StrepTactin to acquire a recognition signal was blocked by the excess of Strep-tagII in solution. This proved that the recognition signals shown in Figure 2B arose from specific interaction between Strep-Tactin coupled to the AFM tip and Strep-tagII on the surface of the cocrystallized S-layer lattice. Subsequently, a smaller scan area on the surface of the cocrystallization of rSbpA-Strep-tagII and SbpA was imaged to increase the lateral resolution. Figure 3A and B show the simultaneously acquired topography and recognition images with a scan size of 216 × 216 nm2. In order to precisely localize the position of Strep-tagII, a “center of mass” approach was used (see Experimental Section). Even though the diameter of the recognition spot was in the order of 2× the length of PEG linker and protein (∼7 nm), the position of the recognition spots was determined with accuracy below 1 nm. By comparing the positions of the recognition sites 4313
Figure 1. (A) Topography image of S-layer protein rSbpA-Strep-tagII recrystallized on a silicon chip. (B) Schematic drawing of StrepTactin conjugated to the AFM tip. The crosslinker NHS-PEG7-S-S-PEG7-NHS was covalently bound to the ethanolamine coated tip through the NHS-ester end. TCEP cut the disulfide bond and Strep-Tactin-SMSS was conjugated to the SH end. (C) Typical force-distance cycle for a recognition event between Strep-Tactin and Strep-tagII. Force-distance cycle without recognition event (inset). (D) Distribution of the unbinding force for the interaction of Strep-Tactin and Strep-tagII in the S-layer protein lattice of rSbpA-Strep-tagII in the presence (dotted) and absence (solid) of free Strep-tag II.
calculated from the recognition image recorded in the opposite scanning direction, a root mean squared distance of 1.5 nm was calculated. This deviation can be explained by the asymmetry introduced by the cantilever movement and the position of Strep-Tactin on the AFM tip. In order to reduce this effect of the scanning direction, the position of the Strep-tagII was identified as the mean position of 4314
corresponding recognition sites calculated for both scanning directions. By overlaying the topography image of StreptagII (Figure 3A) with its recognition image (Figure 3B), 88% of the position of Strep-tagII was found to be located on the corner of the square lattice. Figure 3D shows the schematic drawing of the cocrystallization of rSbpA-StreptagII and wild-type SbpA representing the square area in Nano Lett., Vol. 8, No. 12, 2008
Figure 2. Specificity of recognition imaging. Topography (A) and recognition (B) images of the S-layer proteins rSbpA-Strep-tagII and wild-type SbpA cocrystallized (in a molar ratio of 1:7) on a silicon chip. Topographic (C) and recognition (D) images acquired after Strep-Tactin on the AFM tip was blocked by adding free Strep-tagII.
Figure 3C. The red spot indicates where the Strep-tagII is located in correspondence to the dark spots of the recognition image. The crystalline lattice surface of rSbpA-Strep-tagII with the exposed functional group provides an excellent biotemplate for controlled immobilization of biologically active molecules. Here, two different S-layer lattices were investigated in this aspect: an S-layer lattice formed by rSbpAStrep-tagII alone, and an S-layer containing a mixture of rSbpA-Strep-tagII and wild-type SbpA, cocrystallized at a molar ratio of 1:7. Figure 4A and C show the topographical images of S-layer nanoarrays of rSbpA-Strep-tagII and the cocrystallized rSbpA-Strep-tagII and wild-type SbpA, reNano Lett., Vol. 8, No. 12, 2008
spectively. The square lattice symmetry was observed in both S-layer nanoarrays. In order to certify the accessibility of Strep-tagII, 50 µL of free Strep-Tactin (0.4 mg/ml) was added to the crystalline lattice of the cocrystallized rSbpA-Strep-tagII and wild-type SbpA directly after recording figure 4A. It was shown that the Strep-Tactin molecules only bound to a limited number of S-layer proteins (Figure 4B). These results indicated that the cocrystallization of rSbpA-Strep-tagII and wild-type SbpA disturbed neither the self-assembling properties of the S-layer proteins nor the functionality of the incorporated Strep-tags. Tagged protein subunits were randomly mixed with untagged ones and Strep-Tactin was specifically bound 4315
Figure 3. Topography (A) and recognition (B) images of the S-layer proteins rSbpA-Strep-tagII and SbpA (in a molar ratio of 1:7) cocrystallized on a silicon chip. (C) Superimposition of a recognition map of Strep-tagII onto the corresponding topography image. (D) Schematic drawing of cocrystallized rSbpA-Strep-tagII and wild-type SbpA.
by the Strep-tagII moieties. This evidenced their functional accessibility, which is in good agreement with the results of TREC experiments shown above (Figure 2). Binding of Strep-Tactin was also clearly resolved on top of a complete (100%) rSbpA-Strep-tagII lattice surface (Figure 4D). The density of Strep-Tactin on the pure rSbpA-Strep-tagII lattice was significantly higher than that on the nanoarray surface formed from cocrystallized S-layer (Figure 4B). The arrangement of Strep-Tactin reflected the symmetry of the underlying Strep-tagII binding sites (Figure 4D). In conclusion, the reassembly of genetically functionalized and stable 2D protein nanoarrays on solid supports has been demonstrated in the present study. The functional groups on the surface of the S-layer lattice were exposed on the surface and well accessible to the corresponding ligand. Using simultaneously topography and recognition images, the location of the functional groups in the protein nanoarrays could be monitored with nanoscale resolution. Our experiments demonstrate the use of self-assembled S-layer monolayers as biotemplate for building highly ordered molecular blocks, suitable as a platform for immobilizing molecules with controlled density and spacing. This opens the pos4316
sibility that nanoarrays formed by self-assembly of recombinant S-layer proteins comprising functional domains (e.g., metal binding sequences) could serve as a useful biotemplate for applications in nanotechnology. Experimental Section. Growth of Lysinibacillus sphaericus CCM 2177 and isolation of the wild-type S-layer protein SbpA. Ly. sphaericus CCM 2177 was grown in continuous culture as previously described by Ilk et al.28 Isolation of the S-layer protein wild-type SbpA was done according to Egelseer et al.30 Cloning of the Gene Encoding rSbpA-Strep-tagII. The gene encoding the SbpA derivative rSbpA-Strep-tag II, carrying the Strep-tag II at the C-terminus, was cloned as described before.10,15 The gene encoding rSbpA-Strep-tag II was amplified by PCR from chromosomal DNA of Ly. sphaericus CCM 2177 using the oligonucleotide primer sbpA37 (forward primer) [5′-CGG AAT TCC ATG GCG CAA GTA AAC GAC TAT AAC AAA ATC-3′] and SbpAII/5 (reverse primer) [5′-GA CCG CTC GAG TTA GCA ACC ACC ACC ACC ACC TTC TGA ATA TGC AGT AGT TGC TGC-3′]. The primer SbpAII/5 introduced the sequence of Strep-tag II Nano Lett., Vol. 8, No. 12, 2008
Figure 4. Topographical images of the S-layer protein rSbpA-Strep-tagII and wild-type SbpA (in a molar ratio of 1:7) cocrystallized on a silicon chip in the absence (A) and in the presence (B) of Strep-Tactin. Topographical images of the S-layer proteins rSbpA-Strep-tagII recrystallized on a silicon chip in the absence (C) and in the presence (D) of Strep-Tactin.
(double underlined) at the 3′end of the coding sequence of sbpA. For cloning NcoI and XhoI restriction sites were introduced during PCR at the 5′and 3′end, respectively. For cloning the gel-purified PCR fragment was inserted into the corresponding restriction sites of the plasmid pET28a which was established in Escherichia coli TG1.10 Expression of rSbpA-Strep-tagII. Heterologous expression of the gene encoding rSbpA-Strep-tagII in E. coli HMS(174)DE3 cultures was done as described in Ilk et al.10 Isolation and Purification of rSbpA-Strep-tagII. Isolation of rSbpA-Strep-tagII was performed as described by Jarosch et al.31 For extraction of the S-layer fusion protein, the pellets were resuspended in 4 mL of 5 M guanidine hydrochloride (GHCl) in 50 mM Tris-HCl buffer (pH 7.2), and the suspensions were centrifuged at 36 000× g for 30 min at 4 °C. The supernatants containing rSbpA-Strep-tagII was diluted 1:2.5 with 50 mM Tris-HCl buffer (pH 7.2) and Nano Lett., Vol. 8, No. 12, 2008
filtered through a 0.45 µm RC-membrane (Minisart RC 25). The clear filtrate was finally subjected to gel permeation chromatography (GPC) using a Superdex 200 column (Pharmacia; Upsala, Sweden) equilibrated in 2 M GHCl in 50 mM Tris-HCl/150 mM NaCl buffer (pH 7.2). Fractions containing rSbpA-Strep-tagII were pooled, dialysed against bidistilled water for 18 h at 4 °C, lyophilized, and stored at -20 °C. The purity of rSbpA-Strep-tagII was finally checked by SDS-PAGE. Protein Recrystallization of S-layer proteins. One milligram of lyophilized protein (rSbpA-Strep-tagII and wildtype SbpA) was dissolved in 5 M GHCl in 50 mM TrisHCl (pH 7.2) and dialyzed against Milli-Q water for 3 h. After centrifugation, the protein solution was diluted to 0.1 mg/ml with 0.1 mM CaCl2, 0.5 mM Tris buffer (pH 9). Silicon wafers (Si-Mat Silicon Materials, Germany) were cut into 8 × 8 mm pieces. The silicon pieces were cleaned with 4317
H2SO4/H2O2 (7:1) for 30 min and washed with Milli-Q water for three times. Cleaned silicon wafers were incubated with S-layer protein solution for 4 h, washed with Milli-Q water, and stored in Tris buffer (0.5 mM Tris, 150 mM NaCl, and pH 7.5) at 4 °C. Coupling Strep-Tactin with SMCC. Strep-Tactin-SMCC was prepared by reacting 1 mg/ml Strep-Tactin (IBA) in 0.5 mL buffer A (containing 100 mM NaCl, 50 mM NaH2PO4, 1 mM EDTA, pH 7.5 adjusted with NaOH) with 1.3 mM SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane1-carboxylate, added from a 66 mM stock solution in DMSO) (Aldrich) for 30 min, followed by removal of unbound reagent by gel filtration in buffer A on a PD-10 column (GE Healthcare), which gave a final Strep-Tactin-SMCC concentration of 0.635 mg/ml. Functionalization of AFM Tips with Strep-Tactin. After cleaning with ethanol and chloroform, AFM tips (Si3N4, Veeco, U.S.A.) were incubated with 0.55 g/mL ethanolamine hydrochloride (Sigma) in DMSO overnight at room temperature, yielding functional amino-groups on the tip surface.24 Attachment of the polyethylene glycol (PEG) linkers (NHSPEG7-S-S-PEG7-NHS, Polypure, Norway) was accomplished by incubating the tip for 2 h with 1 mg/mL of PEG linker in chloroform containing 0.5% (v/v) of triethylamine. After rinsing with chloroform, the tip was stored in argon-filled desiccators for no more than 4 days. For coupling of StrepTactin, AFM tips with PEG linkers were immersed in buffer B (100 mM NaCl, 300 mM NaH2PO4, 1 mM EDTA, pH 7.5 adjusted with NaOH) containing 200 µg/ml Strep-TactinSMCC and 2 mM TCEP (tris (2-Carboxyethyl) phosphine hydrochloride) for 2 h, washed in PBS (1.8 mM KH2PO4, 10.1 mM Na2HPO4, 2.7 mM KCl and 140 mM NaCl, pH 7.5) three times and stored in PBS at 4 °C until used (up to 3 days). AFM Measurements. AFM measurements were performed on a Pico Plus setup (Agilent Technologies, Chandler, USA). Magnetically coated AFM cantilevers with nominal spring constant of 0.1 N/m (Agilent Technologies, Chandler, USA) were used for MAC mode imaging. The measurement frequency was set to 20% below the resonance frequency. All measurements were carried out in a buffer containing 0.5 mM Tris and 150 mM NaCl (pH adjusted to 7.5 with HCl). The scan speed for imaging was 1 line/s at 512 data points per line. AFM tips without magnetic coating (Veeco, Santa Barbara) were used for contact single-molecule force spectroscopy. The spring constants of the cantilevers were determined by the thermal-noise mode.32 The Strep-Tactinmodified AFM cantilevers were blocked by addition of 36 µg/ml free Strep-tagII (IBA). Localizing the Position of Strep-tagII by Making Use of the Recognition Image. A manually selected recognition spot was cut out using a 31 × 31 pixels mask. Within this mask, pixels corresponding to the recognition spot were identified by applying a threshold algorithm. The threshold was set to be two thirds of the pixel value range above the average pixel value. The position was estimated using a center of mass approach: 4318
∑ br s
k k
b) r
k
(1)
∑s
k
k
Here b rk) (xk,yk) are the coordinates of the k-th pixel and sk is the corresponding recognition signal, with subtracted background. The sum is executed over all pixels of a recognition spot. This can be treated as deriving a expectation value, therefore the accuracy for determining b¯r was given by sqrt(var b/n), r where n is the total number of pixels in the recognition spot and var b¯r is the variance calculated according to
∑ (br - b)r s 2
k
varb r)
k
k
∑s
(2)
k
k
Acknowledgment. This work was supported by the FP6 EC-STREP Project NASSAP 13523, the U.S. Air Force Office of Scientific Research (AFOSR) Project FA9550-070313, and the Human Science Frontier Program of the European Union (HFSP project RGP0053). References (1) Sleytr, U. B.; Huber, C.; Ilk, N.; Pum, D.; Schuster, B.; Egelseer, E. M. FEMS Microbiol. Lett. 2007, 267, 131–144. (2) Sleytr, U. B.; Sa´ra, M.; Pum, D.; Schuster, B. Prog. Surf. Sci. 2001, 68, 231–278. (3) Sleytr, U. B.; Beveridge, T. J. Trends Microbiol. 1999, 7, 253–260. (4) Sleytr, U. B.; Messner, P.; Pum, D.; Sa´ra, M. Angew. Chem., Int. Ed. 1999, 38, 1034–1054. (5) Sleytr, U. B.; Egelseer, E. M.; Ilk, N.; Pum, D.; Schuster, B. FEBS J. 2007, 274, 323–334. (6) Goodsell, D. S. In Bionanotechnology, Lessons from Nature; Sons, J. W., Ed.; 2004. (7) Sleytr, U. B.; Sa´ra, M.; Pum, D.; Schuster, B. Crystalline bacterial cell surface layers (S-layers): a versatile self-assembly system. In Supramolecular Polymers, 2nd Ed.; Ciferri, A., Ed.; CRC Press: Boca Raton, 2005; pp 583-616. (8) Whitesides, G. M.; Mathias, J. P.; Seto, C. T. Science 1991, 254, 1312– 1319. (9) Huber, C.; Ilk, N.; Runzler, D.; Egelseer, E. M.; Weigert, S.; Sleytr, U. B.; Sa´ra, M. Mol. Microbiol. 2005, 55, 197–205. (10) Ilk, N.; Vo¨llenkle, C.; Egelseer, E. M.; Breitwieser, A.; Sleytr, U. B.; Sa´ra, M. Appl. EnViron. Microbiol. 2002, 68, 3251–3260. (11) Moll, D.; Huber, C.; Schlegel, B.; Pum, D.; Sleytr, U. B.; Sa´ra, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 14646–14651. (12) Pleschberger, M.; Neubauer, A.; Egelseer, E. M.; Weigert, S.; Lindner, B.; Sleytr, U. B.; Muyldermans, S.; Sara, M. Bioconjug. Chem. 2003, 14, 440–448. (13) Pleschberger, M.; Saerens, D.; Weigert, S.; Sleytr, U. B.; Muyldermans, S.; Sara, M.; Egelseer, E. M. Bioconjug. Chem. 2004, 15, 664–671. (14) Tschiggerl, H.; Breitwieser, A.; de Roo, G.; Verwoerd, T.; Schaffer, C.; Sleytr, U. B. J. Biotechnol. 2008, 133, 403–411. (15) Tang, J.; Ebner, A.; Ilk, N.; Lichtblau, H.; Huber, C.; Zhu, R.; Pum, D.; Leitner, M.; Pastushenko, V.; Gruber, H. J.; Sleytr, U. B.; Hinterdorfer, P. Langmuir 2008, 24, 1324–1329. (16) Kienberger, F.; Mueller, H.; Pastushenko, V.; Hinterdorfer, P. EMBO Rep. 2004, 5, 579–583. (17) Scheuring, S.; Ringler, P.; Borgnia, M.; Stahlberg, H.; Muller, D. J.; Agre, P.; Engel, A. EMBO J. 1999, 18, 4981–4987. (18) Hinterdorfer, P.; Dufrene, Y. F. Nat. Methods 2006, 3, 347–355. (19) Wang, H.; Bash, R.; Lindsay, S. M.; Lohr, D. Biophys. J. 2005, 89, 3386–3398. (20) Ebner, A.; Kienberger, F.; Kada, G.; Stroh, C. M.; Geretschlager, M.; Kamruzzahan, A. S.; Wildling, L.; Johnson, W. T.; Ashcroft, B.; Nelson, J.; Lindsay, S. M.; Gruber, H. J.; Hinterdorfer, P. Chem. Phys. Chem. 2005, 6, 897–900. Nano Lett., Vol. 8, No. 12, 2008
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