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Langmuir 2007, 23, 11134-11138
Oriented Purple Membrane Monolayers Covalently Attached to Gold by Multiple Thiole Linkages Analyzed by Single Molecule Force Spectroscopy Michael Schranz,† Frank Noll,† and Norbert Hampp*,†,‡ Faculty of Chemistry and Materials Sciences Center, UniVersity of Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany ReceiVed July 4, 2007. In Final Form: August 10, 2007 Highly oriented monolayers of bacteriorhodopsin (BR) in purple membrane (PM) form are obtained by the reaction of BR-Q3C, where a cysteine was introduced into the N-terminal region, with a gold surface. Single molecule force spectroscopy was used to show that about 50% of the BRs are covalently bound to the surface. The linkage between the cysteine and the gold causes an additional characteristic peak in the force-distance curves to appear. Because several thousand cysteine-gold bonds exist between each PM patch and the surface, the PM is irreversibly bound. Such oriented PM monolayers may serve as an interface between metal surfaces and biomaterials, which may be linked to the PM surface chemically. Photoelectric applications of BR will benefit from the high degree of orientation obtained by this method.
Introduction Bacteriorhodopsin (BR), a photochromic retinal protein from the cell membrane of Halobacterium salinarum, has been studied for more than two decades as a material for technical applications.1-4 BR is isolated from H. salinarum in the form of purple membrane (PM) patches consisting of BR and lipids only. The BR assembles inside the PM into trimers, which are arranged in a 2D hexagonal crystalline lattice. In Figure 1, the hexagonal lattice of BR-Q3C patches immobilized on gold is shown. BR’s seven transmembrane R-helical segments (A-G) enclose a binding pocket for the all-trans retinylidene chromophore, bound via a protonated Schiff base to lysine K216.5 The photocycle of BR has been studied in detail.6,7 Quite a number of possible technical applications would benefit from a high degree of orientation of the PM patches (e.g., photoelectric applications). Per single BR layer, a photovoltage of up to 250 mV is generated upon illumination.2 A high degree of orientation of the PMs is necessary because counter-oriented PMs cancel out the whole effect. Another interesting application of oriented PMs is interface formation between metals and biocomponents8,9 or nanocrystals.10 The BR molecules inside the PM form a nanoscaled pattern of binding sites for biocomponents. The spacing of the attachment sites matches the size of biomaterials such as enzymes. * 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) Birge, R. R.; Gillespie, N. B.; Izaguirre, E. W.; Kusnetzow, A.; Lawrence, A. F.; Singh, D.; Song, Q. W.; Schmidt, E.; Stuart, J. A.; Seetharaman, S.; Wise, K. J. J. Phys. Chem. B 1999, 103, 10746. (2) Hampp, N. A.; Oesterhelt, D. In Nanobiotechnology: Concepts, Applications and PerspectiVes; Mirkin, C., Niemeyer, C., Eds.; Wiley-VCH: Weinheim, Germany, 2004; p 146. (3) Hampp, N. Appl. Microbiol. Biotechnol. 2000, 53, 633. (4) Hampp, N.; Silber, A. Pure Appl. Chem. 1996, 68, 1361. (5) Luecke, H.; Schobert, B.; Richter, H.-T.; Cartailler, J.-P.; Lanyi, J. K. J. Mol. Biol. 1999, 291, 899. (6) Lanyi, J. K. Biochim. Biophys. Acta 2006, 1757, 1012. (7) Lanyi, J. K. Annu. ReV. Physiol. 2004, 66, 665. (8) Fischer, T.; Hampp, N. A. Soft Matter 2007, 3, 707. (9) Seitz, A.; Schneider, T.; Pasternack, R.; Fuchsbauer, H.-L.; Hampp, N. Biomacromolecules 2001, 2, 233. (10) Yu, S. M.; Mo, X.; Krebs, M. P. MRS Symp. Proc. 2003, 735, 21.
Figure 1. Hexagonal crystalline structure of BR-Q3C. (Inset) BRQ3C patches immobilized on gold.
Many techniques to achieve highly oriented films of PM have been explored in the past, such as Langmuir-Blodgett depositon,11 preferential orientation at interfaces,12 and electric field sedimentation,13 but not all of these techniques achieve a high degree of orientation. The only dependable method so far is the coating of a surface with monoclonal antibodies against the cytoplasmic or extracellular side of BR,14 but this method is delicate and time-consuming. A promising, easier route to highly oriented PM monolayers is the use of genetically engineered BR variants, whose production has become routine. Wild-type BR (BR-WT) does not contain any cysteines. Genetic engineering enables one to obtain a cysteine located in the extramembraneous parts of the PM either on the cytoplasmic or the extracelluar side. As a result, the orientation (11) Miyasaka, T.; Koyama, K. Thin Solid Films 1992, 210-211, 146. (12) Fisher, K. A.; Yanagimoto, K.; Stoeckenius, W. J. Cell Biol. 1978, 77, 611. (13) Keszthelyi, L. Biochim. Biophys. Acta 1980, 598, 429. (14) Koyama, K.; Yamaguchi, N.; Miyasaka, T. Science 1994, 265, 762.
10.1021/la7019928 CCC: $37.00 © 2007 American Chemical Society Published on Web 09/22/2007
Oriented Purple Membrane Monolayers
Figure 2. Scheme of a purple membrane (PM), which consists of bacteriorhodopsin (BR, purple) and lipids (gray) only, immobilized onto gold by multiple thiol-gold bonds (green). Oriented PMs are interesting (i) as templates for the immobilization of biocomponents (blue), (ii) because of their photoelectric properties, and (iii) as combinations of both.
of PM monolayers on gold may be determined by the modification introduced. The BR variant used here is Q3C, where glutamine (Q) in position 3 is replaced by cysteine (C). Therefore, a binding site in the N-terminal region is introduced that causes PM to bind to a gold surface selectively on the extracellular side. Immobilization of proteins to gold surfaces via a cysteine is widely used (e.g., ref 15), but here thousands of gold-sulfur bonds coupled by the crystalline structure of PM attach each PM to the surface almost irreversibly (Figure 2). This binding event may be analyzed by single molecule force spectroscopy, a method that has become a powerful tool in manipulating single proteins and in obtaining structural information.16-18 Experimental Section Bacteriorhodopsin and Sample Preparation. The BR-Q3C variant material was a gift from Dieter Oesterhelt’s laboratory. It was purified following a standard procedure.19 BR-Q3C was protected from oxidation by storing it in a solution containing excess mercaptoethanol. To deprotect it, first the mercaptoethanol was removed by washing two times with water, an excess of a phosphine buffer (1 mM Tris(2-carboxyethyl)phosphine (Sigma-Aldrich, Steinheim, Germany), 20 mM sodium acetate, pH 4.5) was added to the PM suspension to reduce disulfides, and the PM-phosphine suspension was incubated for 30 min at 40 °C. The resulting material was washed twice in a centrifuge (15 min, 12 000 rpm, Biofuge 13, Heraeus, Hanau, Germany), and the supernatant was discarded. The pellet was dissolved in a suspension buffer (150 mM KCl, 10 mM Tris-HCl, pH 8.2) to a final absorbance of 0.2 to 0.3 at 570 nm. The deprotected PM solution (10 µL) was cast immediately together with 50 µL of incubation buffer (300 mM KCl, 10 mM Tris-HCl, pH 7.820) onto a gold surface (TSG is template-stripped gold21). After 30 min of incubation, excess material was removed by extensive washing with incubation buffer. AFM Imaging and Single Molecule Force Spectroscopy. AFM imaging was performed in liquid (incubation buffer) on a Nanoscope IV system equipped with a PicoForce modul (Veeco, Santa Barbara, CA). Pyramidal, oxide-sharpened Si3N4 tips attached to a V-shaped substrate (Olympus, Tokyo, Japan) were used for imaging and force spectroscopy. The spring constant of the cantilevers was measured (15) Ros, R.; Schwesinger, F.; Anselmetti, D.; Kubon, M.; Scha¨fer, R.; Plu¨ckthun, A.; Tiefenauer, L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7402. (16) Oesterhelt, F.; Oesterhelt, D.; Pfeiffer, M.; Engel, A.; Gaub, H. E.; Mu¨ller, D. J. Science 2000, 288, 143. (17) Mu¨ller, D. J.; Kessler, M.; Oesterhelt, F.; Mo¨ller, C.; Oesterhelt, D.; Gaub, H. E. Biophys. J. 2002, 83, 3578. (18) Kessler, M.; Gaub, H. E. Structure 2006, 14, 521. (19) Oesterhelt, D.; Stoeckenius, W. Methods Enzymol. 1974, 31, 667. (20) Mu¨ller, D. J.; Fotiadis, D.; Scheuring, S.; Mu¨ller, S. A.; Engel, A. Biophys. J. 1999, 76, 1101. (21) Wagner, P.; Hegner, M.; Gu¨ntherodt, H.-J.; Semenza, G. Langmuir 1995, 11, 3867.
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Figure 3. Scheme of a BR-Q3C incorporated in a PM where the cysteine in position 3 is covalently attached to the gold surface. The amino acids of the BR molecules are arranged in seven transmembrane R-helices. The cysteine in the N-terminal region of the protein is marked in green. Schematically shown is an AFM tip interacting with the C-terminal region of the protein. in solution using thermal fluctuation analysis.22 All cantilevers had a spring constant of 0.08 N/m within the uncertainty of this method (10%). Before pulling single BR molecules off of the PM sheet, PM patches were scanned, and the tip was positioned over a patch. Then the tip was attached to the sample and pressed onto it for 0.2 to 0.3 s at a force of 1.0-1.5 nN. The retraction velocity was about 1.1 µm/s. Attachment of a protein to the tip is detected by deflection of the cantilever. The force-distance curve obtained represents the unfolding of a single BR molecule. In less than 2% of the experiments, a binding event between the tip and BR was observed. One-third of the resulting curves showed four main peaks, indicating the unfolding of a single BR molecule. Mu¨ller et al.17 demonstrated that a cytoplasmic force-distance curve with an overall length of more than 50 nm can be measured only in cases where the tip is attached to the C terminus of the protein (Figure 3). Tip attachments to any of the amino acid loops connecting the R-helical domains can be excluded for such curves. The length of the stretched amino acid chain was determined using the contour length of a wormlike chain (WLC) fit23,24 with a persistence length of 0.4 nm.25 By dividing the contour length of the fit curves by the monomer length (0.36 nm), the number of amino acids (aa) that were stretched yielding a corresponding peak in the force spectrum was calculated. The first main peak shifts from force spectrum to force spectrum and cannot be analyzed like the other main peaks. Peaks 2-4 occur at chain lengths of 88, 148, and 219 aa. The force curves measured with BR-Q3C look slightly different. The rupture forces of unfolding experiments are rate-dependent.26 Therefore, only relative force differences are discussed rather than absolute forces.
Results and Discussion BR-Q3C patches oriented with their extracellular sides toward the gold surface may form numerous sulfur-gold bonds and become covalently attached to the gold surface. In the case in which the cytoplasmic side is oriented toward the gold surface, no binding is expected. During sample preparation and washing steps, all unbound material is removed from the gold surface. For PMs oriented with their extracellular side toward the gold surface, some cysteine residues may stay unbound because they either were oxidized by oxygen from the air or are still protected (22) Hutter, J. L.; Bechhoefer, J. ReV. Sci. Instrum. 1993, 64, 1868. (23) Bustamante, C.; Marko, J. F.; Siggia, E. D.; Smith, S. Science 1994, 265, 1599. (24) Marko, J. F.; Siggia, E. D. Macromolecules 1995, 28, 8759. (25) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109. (26) Janovjak, H.; Struckmeier, J.; Hubain, M.; Kedrov, A.; Kessler, M.; Mu¨ller, D. J. Structure 2004, 12, 871.
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Figure 4. Force-distance curves obtained from BR-Q3C. (A) Curves with four peaks (1-4) corresponding to BRs showing no sulfur-gold binding (n ) 10). (B) Curves with five peaks. Peaks 1-4 are identical to those in A but show an additional fifth peak (83 nm) (n ) 9). The rupture force of the fifth peak is higher than that of the fourth peak. (C) Close-up of the fourth and fifth peaks shown in B. Two peaks can be distinguished easily. (D) Curves where the fourth peak is missing but only the fifth peak at a longer contour length showing an increased rupture force is observed (n ) 15). The curves shown in B and D represent BR covalently bound to the gold surface.
by mercaptoethanol or because the distance between cysteine and the surface is too large to form a covalent bond. This may happen because even TSG substrates show a certain surface roughness. BR unfolds sequentially when pulled off of PM.16,17 Pairwise unfolding of the R-helices leads to a characteristic force-distance curve pattern. The first peak represents the stretching of the C-terminal end, and the second, third, and fourth are due to the pairwise unfolding and stretching of the F & G, D & E, and B & C helices. The decrease in the force after the fourth peak may be interpreted as the point where either the protein is finally pulled off of the membrane (helix A) or the tip is detached from the protein. Oesterhelt et al. demonstrated by AFM imaging with molecular resolution16 that in most cases the protein is pulled off of the PM. The scheme for BR-Q3C being covalently attached to a gold surface is shown in Figure 3. In the case in which the AFM tip was bound to the C-terminus of the protein, three different forcedistance curves were obtained, differing in the number of peaks and in the force of the last peak (Figure 4). Figure 4A shows the case of unbound BR-Q3C molecules. Four main peaks appear at the same positions as obtained with wild-type BR deposited on mica or gold (Figure 5). In all cases where the cysteine is bound to the gold surface, a new peak at a larger contour length than that found for the usual four main peaks is observed because of the new barrier at the N-terminal end of the protein. This new peak may be interpreted as the unfolding of helix A, which mostly is not
observed with wild-type BR. Two different types of forcedistance curves with the additional peak are observed (Figure 4B,D). In Figure 4B, peaks 1-4 are in the positions found for unbound BR-Q3C or BR-WT (Figure 5). Instead of a full decrease in the force after the fourth peak, only a small sharp reduction in force is observed, and then a fifth peak develops from there corresponding to a length of 83 nm or 231 aa. A close-up of this region of Figure 4B is shown in Figure 4C to demonstrate in more detail the appearance of the new barrier that we assign to the covalent attachment of the protein to the surface, which results in the additional peak and its higher rupture force. The additional barrier is located on the extracellular side. Because the zero point of the WLC fits is located on the cytoplasmic side of the PM, the contour length of the fitted WLC curves is smaller than the length of the stretched protein. To calculate the exact length of the stretched amino acid chain, the distance between the cytoplasmic surface and the barrier (about 4 nm ) 11 aa) has to be added to the number of stretched amino acids given by the WLC curve.17 Because the introduced cysteine is in position 3, the stretched amino acid chain should be 245 aa long (248 aa - 3 aa). Considering the position of the barrier, the contour length of the WLC-fit should be about 234 aa (245 aa - 11 aa). The contour lengths of the fit curves obtained are about 231 aa. The second type of curve observed is shown in Figure 4D. These force-distance curves have only one peak corresponding to the additional barrier at 231 aa (fifth peak in Figure 4B) and no peak or only a very small peak at 217 aa. Almost half of the measured Q3C force curves show this progression. The missing
Oriented Purple Membrane Monolayers
Figure 5. Force-distance curves measured for BR-WT (A) on gold and (B) on mica.
fourth main peak indicates that helices A-C are pulled off of the membrane simultaneously, whereas the additional peak (fifth peak) indicates that the BR monomer is bound to the gold surface covalently. The average rupture force of the fifth peak measured on curves showing a fourth peak or no peak is higher than the fourth peak of unbound BR-Q3C (Figure 4A). To verify that the measured forces do not arise from effects other than the interaction between the cysteine group of BRQ3C and the gold surface, force curves of BR-WT deposited on TSG surfaces (Figure 5A) and mica (Figure 5B) were measured as a reference. The positions of the main peaks were in the same range as published16-18 for BR-WT on mica. Not a single BRWT force-distance curve was found that showed an additional peak. Furthermore, the rupture force of the fourth peak amounts to significantly less than the rupture force of the additional fifth peak of BR-Q3C in all cases. The average forces of all fitted peaks are summarized in Table 1. In Figure 6A, force curves from BR-WT and BR-Q3C curves showing a covalent binding event measured on gold are compared. The positions of the fitted peaks are marked in a scheme of the BR secondary structure (Figure 6B). BR-WT and unbound BR-Q3C force curves comprising three peaks as well as those comprising four peaks are obtained. This means that in some cases helices A-C are extracted simultaneously and in some cases first B-C and then A are extracted. Because the distance between the second and third peak is the same in both cases, it can be concluded that these shorter curves correspond to BR monomers that are attached with their C-terminal end to the tip. In the latter version, where a barrier corresponding to the fourth peak in Figure 6 is observed, the introduced cysteine-gold bond leads to a fifth peak that represents
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Figure 6. (A) Superposition of BR-WT force curves (black dots, n ) 30) and BR-Q3C curves showing a peak at a contour length of 231 aa (orange dots, n ) 15). (B) Positions of the barriers shown in a scheme of the BR secondary structure. All positions are marked by numbers corresponding to those of the force curve. Table 1. Summary of Contour Lengths and Forces Measured for BR-Q3C Deposited on Gold and for BR-WT Deposited on Gold or Mica rupture force no. of (pN) curves
peak number
contour length (aa/nm)
Q3C (Figure 4A)
2 3 4
88 aa/32 nm 147 aa/53 nm 217 aa /78 nm
160 101 31
100 87 94
10
Q3C (Figure 4B)
2 3 4 5
88 aa/ 32 nm 150 aa/54 nm 217 aa/78 nm 231aa/83 nm
160 98 31 6
95 94 84 100
9
Q3C (Figure 4D)
2 3 5
88 aa/32 nm 150 aa /54 nm 231 aa/83 nm
160 98 6
85 83 106
15
WT on gold (Figure 5A)
2 3 4
88 aa/32 nm 146 aa/53 nm 214 aa/77 nm
160 102 34
127 90 80
28
WT on mica (Figure 5B)
2 3 4
88 aa/32 nm 147 aa/53 nm 215 aa/77 nm
160 101 33
126 85 82
30
amino acid pos. (aa)
the pulling of helix A. In the case where only three peaks are observed in BR-WT (i.e., A-C are extracted simultaneously), the fourth peak is missing, but the fifth peak is observed, which again refers to the pulling of helix A. The reason for the distribution between three-peak and four-peak curves is unclear. A possible explanation might be that the extraction of the first BR from a trimer leads to change in the stability of the remaining two BRs, which may result in two different types of force curves.
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Kessler et al.18 and Mu¨ller et al.17 have described a side peak at a contour length of about 242 aa (the position of the barrier is already added here) for BR-WT deposited on mica. Whereas the measured rupture force described by Kessler et al. decreases because there are no strong interactions between BR-WT and mica, the results presented here show a significant increase in the force from the fourth to the fifth peak (about 19%, see Table 1). Mu¨ller et al.17 found only a slightly increased force for the side peak at 242 aa (about 6%). The obviously increased force measured here and the more frequent appearance of this peak are interpreted as evidence of a gold-cysteine binding event. Of course the results of this work are not statistical satisfying because of the limited amount of data. From the measured forcedistance curves, we derive a binding rate of cysteine to the gold surface of about 50% because this amount of force curves showed an interaction leading to an additional peak in the range of 234 aa indicating the unfolding of helix A. Unfortunately, the molecular resolution of BR-Q3C on the TSG substrate was not achieved, so it is not possible to decide directly which side was exposed. (The highest resolution obtained is shown in Figure 1.) All measured force curves fitted to the contour lengths were calculated for the case in which the proteins are pulled off of the cytoplasmic side. Because of the insufficient resolution of the AFM images, it is not clear whether single BR proteins showing a binding event were pulled off of the membrane or the tip was detached from the protein. However, as far as the orientation of the PM may be distinguished on the basis of the properties of the force curves,18 it is concluded that in the
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experiments presented here all of the proteins were pulled off of the cytoplasmic side. The formation of cysteine-gold bonds and those between membrane-embedded BR-S35C and a flat gold substrate has been observed by XPS.27 Covalent bonds between cysteine and gold but also between sulfur cleaved from the methionine end group and gold were observed. Because methionine residues occur on both sides of the PM (extracellular, M65; cytoplasmic, M163), this is not a suitable approach for oriented assemblies of PMs, in contrast to the results presented here. The cysteine of BR-Q3C in the N-terminal region is more easy accessible than all residues mentioned above.
Summary and Conclusions For fundamental studies on the unfolding of BRs from PM, the cysteine linkage allows us to observe the unfolding of helix A. The chemical attachment of PMs via numerous cysteinegold bonds to a metal surface may be a valuable tool for achieving larger oriented monolayers of PMs on a solid support in an easy way. Photovoltaic applications of BR may benefit from such oriented monolayers, but the use of BR as an interface and a template for the immobilization of biomolecules on metal surfaces, without having the often denaturing effect of direct metalbiomolecule contact, may also be interesting. LA7019928 (27) Brizzolara, R. A.; Boyd, J. L.; Tate, A. E. J. Vac. Sci. Technol., A 1997, 15, 773.
