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Functionalized Self-Assembled Monolayers on Ultraflat Gold as Platforms for Single Molecule Force Spectroscopy and Imaging Vamsi K. Yadavalli, Jeffrey G. Forbes, and Kuan Wang* Muscle Proteomics and Nanotechnology Section, Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, DHHS, Bethesda, Maryland 20892 ReceiVed February 1, 2006. In Final Form: May 23, 2006 Single molecule force spectroscopy is a valuable tool for studying unfolding and nanomechanical properties of proteins. The common practice is to stretch proteins from a surface that was dosed to give a reasonable hit rate and to analyze the curves that exhibit the expected characteristics of a single polymer. Whether the surface-bound proteins are indeed single and isolated remains unclear, and the undesirable protein/surface interactions that obscure informative features of the force curves are implicitly assumed to be absent. In this study, mixed self-assembled monolayers (SAMs) consisting of N-hydroxysuccinimide (NHS) and oligoethylene glycol (OEG) terminated thiols on an ultraflat gold surface were used to covalently immobilize proteins via lysine residues. By the optimization of attachment sites via lysine-NHS linkages amidst a protein-resistant layer of the OEG SAM, it was possible to isolate single proteins for study in a controlled fashion. The single protein distribution on the surface is clearly demonstrated by atomic force microscopy (AFM) imaging. The OEG also significantly reduces nonspecific tip-surface interactions between the cantilever and surface. Stretching covalently attached single proteins produces high-quality and reproducible forceextension curves. This experimental strategy is an attractive platform with which to study protein structure, interactions, and nanomechanical properties of single proteins.
Introduction The study of single biomolecules enables the understanding of properties and dynamic behavior that are usually averaged out in ensemble (bulk) experiments. Unique structural or temporal states may therefore be identified and studied toward understanding complex processes or bulk properties. Such research involves studying the nanomechanics,1 conformational or structural changes,2,3 and molecular interactions4,5 of single molecules. Atomic force microscopy (AFM)6 is an ideal tool not just for imaging single molecules7,8 but also for studying molecular interactions and nanomechanics. In particular, force-distance spectroscopy9 has been utilized for a variety of single molecule measurements including molecular rupture forces,10 protein domain unfolding,11,12 and receptor-ligand interactions.5,13-15 * To whom correspondence should be addressed. Telephone: (301) 4964097. Fax: (301) 402-8566. E-mail:
[email protected]. (1) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109-1112. (2) Kageshima, M.; Lantz, M. A.; Jarvis, S. P.; Tokumoto, H.; Takeda, S.; Ptak, A.; Nakamura, C.; Miyake, J. Chem. Phys. Lett. 2001, 343 (1-2), 77-82. (3) Forbes, J. G.; Lorimer, G. H. Science 2000, 288 (5463), 63-64. (4) Weisel, J. W.; Shuman, H.; Litvinov, R. I. Curr. Opin. Struct. Biol. 2003, 19, 227-235. (5) Hinterdorfer, P.; Baumgartner, W.; Gruber, H. J.; Schilcher, K.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3477-3481. (6) Binnig, G.; Quate, C. F.; Gerber, C. Phys. ReV. Lett. 1986, 56 (9), 930933. (7) Stroh, C.; Wang, H.; Bash, R.; Ashcroft, B.; Nelson, J.; Gruber, H.; Lohr, D.; Lindsay, S. M.; Hinterdorfer, P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (34), 12503-12507. (8) Hansma, H.; Kim, K.; Laney, D.; Garcia, R.; Argaman, M.; Allen, M.; Parsons, S. J. Struct. Biol. 1997, 119, 99-108. (9) Frederix, P.; Akiyama, T.; Staufer, U.; Gerber, C.; Fotiadis, D.; Muller, D.; Engel, A. Curr. Opin. Chem. Biol. 2003, 7, 641-647. (10) Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Science 1999, 283, 1727-1730. (11) Rounsevell, R.; Forman, J. R.; Clark, J. Methods 2004, 34, 100-111. (12) Forbes, J. G.; Wang, K. J. Vac. Sci. Technol. 2004, 22 (4), 1-5. (13) Allen, S.; Davies, J.; Davies, M. C.; Dawkes, A. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Biochem. J. 1999, 341, 173-178. (14) Lo, Y. S.; Simons, J.; Beebe, T. P. J. Phys. Chem. B 2002, 106 (38), 9847-9852.
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An important consideration in such experiments has been the immobilization of the molecules involved. These molecules may be coupled to the AFM tip16 or the surface,17 or both,18 while maintaining functionality. Another issue is the minimization of nonspecific adhesion of the tip to the surface.19 In AFM experiments studying proteins on a surface, there is often a large nonspecific adhesive force between the surface and the cantilever tip. This results in a cantilever deflection manifested by the presence of a linear nondelayed retraction curve with the same slope as that of the contact region. A combination of factors such as hydrophobic, electrostatic, or van der Waals interactions may result in tip-surface interactions of magnitudes on the order of binding events between an antibody and antigen or receptor and ligand.20 Such confounding factors make the selection and analysis of force-distance spectra considerably difficult. Traditional approaches to single molecule studies have consisted of applying low concentrations of proteins to a surface followed by force-distance spectroscopy. The presence of a single molecule on the surface is then inferred by fitting the force data assuming the presence of single, isolated molecules. More often than not, the proteins tend to aggregate, form films, or associate on the surface. This results in the vast majority of such traces being representative of multiple molecules. In addition to this lack of control on the surface distribution, the presence of such complexes on the surface exacerbates the tip-surface adhesion that presents confounding factors in the analysis of force curves. (15) Hugel, T.; Seitz, M. Macromol. Rapid Commun. 2001, 22 (13), 9891016. (16) Noy, A.; Vezenov, D. V.; Lieber, C. M. Annu. ReV. Mater. Sci. 1997, 27, 381-421. (17) Wagner, P.; Hegner, M.; Kemen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 2052-2066. (18) Harada, Y.; Kuroda, M.; Ishida, A. Langmuir 2000, 16, 708-715. (19) Amirgoulova, E. V.; Groll, J.; Heyes, C. D.; Ameringer, T.; Rocker, C.; Moller, M.; Nienhaus, G. U. ChemPhysChem 2004, 5, 552-555. (20) Israelachvili, J. Intermolecular and surface forces, 2nd ed.; Academic Press: San Diego, CA, 1992.
This article not subject to U.S. Copyright. Published 2006 by the American Chemical Society Published on Web 07/07/2006
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Typically, these regions of the force-distance curve, as well as the tip-surface force obtained for such a trace, have been ignored or mathematically eliminated by approximating the point of contact for each curve.21 Different groups have presented varied approaches toward dealing with this problem. Best et al.22 proposed criteria to select force-distance curves that would remove subjectivity in the determination of which aspects of the spectra are artifacts and which represent “useful” information, representative of single molecules. Willemsen et al. developed a model to calculate this adhesion force of a tip with a mica surface and attempted to minimize the nonspecific adhesion by imaging in the repulsive regime.23 Brogan et al. investigated the use of surfactants to reduce the nonspecific binding events.24 Chemical approaches for tackling this problem have included the use of flexible polyethylene glycol tethers25 and coating surfaces with dextrans to reduce unwanted interactions.26 The former approach using tethers may further complicate proteinstretching data by introducing an additional flexible component that may present dynamics of its own. The latter approach involving dextran requires coating the cantilever tip to reduce nonspecific interactions. In addition, these approaches have not completely solved the problem of clearly separating single molecules on surfaces. Here, we take a surface chemistry approach toward improving the behavior of the substrate to which the proteins are attached. This is a fundamental approach for eliminating the source of the problem by using self-assembled monolayers (SAMs). SAMs have been long studied for their ability to create precisely controlled and functionalized surfaces for studying interfacial phenomena and biomolecular and cellular interactions.27,28 SAMs of ω-substituted alkanethiols on Au(111) surfaces, in particular, have attracted a great deal of attention due to their ease of preparation, their stability, and their ability to create a broad variety and distribution of surface groups and properties.29,30 Mixed SAMs using binary mixtures of functionalized thiols have been synthesized by coadsorption to a surface from a mixture of thiols.31,32 Molecules of interest may then be immobilized at a single molecule level on this functionalized surface based on the controllable functional groups. This paper presents the first systematic approach, to our knowledge, toward isolating single proteins on surfaces with a view to studying their unfolding and nanomechanics. In addition, we present data showing how problematic nonspecific tip-surface interactions may be avoided by the use of self-assembled monolayers. We demonstrate that a mixed thiol solution with a protein-resistant oligoethylene glycol (OEG) thiol SAM along with sparsely populated N-hydroxysuccinimide (NHS) thiol (21) Baumgartner, W.; Hinterdorfer, P.; Schindler, H. Ultramicroscopy 2000, 82, 85-95. (22) Best, R. B.; Brockwell, D. J.; Toca-Herrera, J. L.; Blake, A. W.; Smith, D. A.; Radford, S. E.; Clarke, J. Anal. Chim. Acta 2003, 479 (1), 87-105. (23) Willemsen, O. H.; Snel, M. M. E.; Kuipers, L.; Figdor, C. G.; Greve, J.; De Grooth, B. G. Biophys. J. 1999, 76 (2), 716-724. (24) Brogan, K. L.; Shin, J. H.; Schoenfisch, M. H. Langmuir 2004, 20, 97299735. (25) Hinterdorfer, P.; Gruber, H. J.; Kienberger, F.; Kada, G.; Riener, C.; Borken, C.; Schindler, H. Colloids Surf., B: Biointerfaces 2002, 23, 115-123. (26) Stevens, M. M.; Allen, S.; Davies, M. C.; Roberts, C. J.; Schacht, E.; Tendler, S. J. B.; VanSteenkiste, S.; Williams, P. M. Langmuir 2002, 18, (17), 6659-6665. (27) Ulman, A. Chem. ReV. 1996, 96 (4), 1533-1554. (28) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. ReV. 2005, 105 (4), 1103-1169. (29) Bertilsson, L.; Liedberg, B. Langmuir 1993, 9, 141-149. (30) 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. (31) Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M. J. Adhes. Sci. Technol. 1992, 6 (12), 1397-1410. (32) Laibinis, P. E.; Nuzzo, R. G.; Whitesides, G. M. J. Phys. Chem. 1992, 96 (12), 5097-5105.
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tethers33 can be used to create a surface for the covalent attachment of single protein molecules. The presence of the oligoethylene glycol prevents the adhesion of the protein of interest to all but the exposed NHS groups on the surface. Surfaces with an oligoethylene glycol (OEG) SAM surface have been demonstrated to resist protein adsorption,34,35 resulting from steric repulsion as well as the exclusion of proteins by a water interface layer created by the helical arrays of the OEG chains. It is also shown that the OEG SAM ensures that there is minimal tip-surface interaction at points where a molecular recognition event does not occur. This approach may be extended further for the creation of surfaces that may be used to covalently immobilize biomolecules for devices and biosensors. Other single molecule techniques such as optical tweezers where nonspecific interactions also present confounding factors36 could also be potentially aided by the use of such functionalized platforms. Experimental Procedures Materials and Instrumentation. (1-Mercaptoundec-11-yl) hexaethylene glycol (OEG), HS-C11- (EG)6OH, and (1-mercaptohexadecanoic acid)-N-succinimidyl ester (NHS-terminated thiol), HSC15COO-NHS, were purchased from Prochimia (Gdansk, Poland). Ethanol (200-proof) was purchased from the Warner-Graham Company (Cockeysville, MD). Epotek 377 glue was purchased from Epoxy Technology (Billerica, MA). Ethanolamine and tetrahydrofuran (THF) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphate-buffered saline (PBS; 155.17 mM NaCl, 1.06 mM KH2PO4, 2.97 mM Na2HPO4, pH 7.4) was obtained from Gibco and sterile filtered prior to use. To minimize contamination, experiments were performed in Teflon beakers and containers (Savillex Corp., Minnetonka, MN). Silicon nitride (Si3N4) microlevers from Veeco and ultrasharp Type I MAC cantilevers from Molecular Imaging (Phoenix, AZ) were used for force measurement and imaging. Proteins. A variety of different proteins of different sizes and shapes were surveyed. A large spherical protein (ferritin), an immunoglobulin G (anti-nebulin SH3 antibody), a small intrinsically disordered nebulin protein fragment (ND66 fragment), and a large filamentous protein (myosin) were investigated. To enhance the attachment of single molecules, all purified proteins were applied to a gel filtration column immediately prior to use and the monodisperse fractions selected. Rabbit skeletal myosin was purified and gel filtered as described.37 Rabbit myosin subfragment S2 was gel filtered on a Superose 6 column in 0.6 M KCl, 20 mM sodium phosphate buffer (pH 7.0). Cloned nebulin ND66 fragments were expressed in E. coli as described earlier.38 ND66 was gel filtered on a Superose 12 column equilibrated with 150 mM NaCl, 5 mM CaCl2, 10 mM imidazole (pH 7.0). Ferritin (Type I from horse spleen) was obtained from Sigma-Aldrich (St. Louis, MO). Immediately prior to use, the ferritin was gel filtered using a Superose 6 column in the same buffer. Nebulin anti-SH3 antibodies (polyclonal IgG) were produced in rabbit against commercially synthesized peptide sequences coupled to a KLH carrier. Serum was affinity purified on a peptide conjugated agarose column, eluted with glycine, and gel filtered prior to use using a Superose 6 column in PBS pH 7.4 (Gutierrez and Wang, unpublished data). Functionalized OEG SAM on Template-Stripped Gold Surfaces. Ultraflat gold surfaces were fabricated using the templatestripping process of Wagner et al.39 Briefly, evaporated gold surfaces (33) Wagner, P.; Kernen, P.; Hegner, M.; Ungewickell, E.; Semenza, G. FEBS Lett. 1994, 356, 267-271. (34) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 1071410721. (35) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20. (36) Stout, A. L. Biophys. J. 2002, 80 (6), 2976-2986. (37) Root, D. D.; Yadavalli, V. K.; Forbes, J. G.; Wang, K. Biophys. J. 2006, 90 (8), 2852-2866. (38) Wang, K.; Knipfer, M.; Huang, Q. Q.; van Heerden, A.; Hsu, L. C.; Gutierrez, G.; Quian, X. L.; Stedman, H. J. Biol. Chem. 1996, 271 (8), 4304-14.
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Figure 1. Covalent immobilization of single proteins on mixed self-assembled monolayer of (1-mercaptohexadecanoic acid)-N-succinimidyl ester and (1-mercaptoundec-11-yl) hexaethylene glycol on ultraflat Au(111) surface. Single antibodies are covalently immobilized at different locations and with varied orientations via the sparsely distributed and exposed NHS groups on the SAM surface. on mica substrates were glued face down using epoxy glue Epotek 377 on Si(100) wafers or glass slides that were piranha (3:1 v/v H2SO4:H2O2) cleaned. Proper care was exercised in handling the extremely corrosive piranha solution. The surfaces were then heat cured in an oven for 1-2 h at 150 °C. The template-stripped gold surfaces were obtained by stripping the mica with tetrahydrofuran. The resulting gold surfaces were then washed with ethanol and dried in a stream of purified N2. The RMS (root-mean-square) roughness of the gold surfaces was measured to be less than 0.5 nm over 5 µm. Thiol SAMs were prepared by the incubation of the freshly prepared gold surfaces in a thiol solution in ethanol for 16-24 h. After incubation, the gold surfaces were rinsed with ethanol, dried in a stream of purified N2, and imaged in the buffer. The molar ratio of the OEG thiol to the NHS thiol was empirically determined by titration, and an approximately 10 000:1 ratio was used to obtain well-separated attachment sites on the gold. This also corresponded to a theoretical estimation assuming a distance between attachment sites of 200 nm, assuming an area per molecule of 21.4 Å2.40 Figure 1 shows a schematic of the mixed SAM surface with NHS groups surrounded by protein-resistant oligoethylene glycol thiol (OEG) as prepared by this method. SAM surfaces with 100% OEG6 thiol and 100% NHS thiol were studied as negative and positive controls for the functionalized surface. In both cases, the functionalized surfaces were incubated with protein in PBS for 1 h. The protein concentration for all experiments was ∼1 µg/mL with a total protein of ∼500 ng. The unreacted NHS groups were then quenched for 1 h in 0.1 M ethanolamine solution at pH 7.4. The surfaces were washed with PBS and imaged in noncontact mode. All experiments were performed at room temperature unless otherwise specified. MAC mode AFM cantilever tips for imaging were cleaned by exposure to ozone in the presence of high-intensity UV light (254 nm) to remove any contamination by organics on the surface, while preserving the magnetic coating on the cantilevers. Softer cantilever tips for force-distance measurements were cleaned in a solution of (39) Wagner, P.; Hegner, M.; Guntherodt, H.-J.; Semenza, G. Langmuir 1995, 11, 3867-3875. (40) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-436.
ethanolic KOH (0.5 M) followed by extensive rinsing in ethanol and water. Cantilevers were calibrated and their force constants measured using the thermal fluctuation method.41 Atomic Force Microscopy: Single Molecule Force Curves and Imaging. For adsorbing the myosin on the gold surface, 190 µL of sterile filtered buffer (0.5 M KCl, 20 mM imidazole, pH 7.0, 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP)) was placed on the freshly prepared SAM and 10 µL of protein solution in the same buffer was carefully injected under the buffer surface and gently mixed. Final concentrations were ∼1 µg/mL. The protein was incubated for 30 min at room temperature (22 °C), and the surface was then rinsed three times with the same buffer to remove any unattached protein. A buffer near physiological ionic strength (0.1 M KCl, 20 mM imidazole, pH 7.0, 1 mM TCEP, pH 7.0) was then added (550 µL), and AFM measurements were immediately taken. A PicoSPM AFM instrument (Molecular Imaging, Phoenix, AZ) in both contact and noncontact (MAC) mode was used for forcedistance and imaging measurements. Soft cantilever tips (nominal force constant ∼0.01 N/m, resonance frequency 7 kHz) were used for the force-distance analysis. Stiffer cantilevers (nominal force constant 0.6-0.95 N/m, resonance frequency 75-105 kHz) were used for imaging in the MAC mode. Freshly cleaned Si3N4 tips were used to measure the presence and magnitude of nonspecific adhesion to clean surfaces. Clean gold, glass, silicon, and mica substrates were investigated as substrates. Epitaxially deposited gold on mica stored under Ar was cleaned in UV/ozone. Glass and polished Si(111) were cleaned immediately prior to use using piranha solution. Surfaces were washed extensively with water and imaged in PBS. Force spectra were obtained by moving the tip in contact with the surface, holding it in place for some time to allow binding to occur and then retracting in a repeated, cyclic manner. Several hundred curves were obtained for each experiment by moving the tip to different points on the surface. The cantilever speed was ∼1 µm/s and the force of contact was ∼1 nN to avoid damaging the surface protein. Pull-off forces were analyzed using Scanning Probe Image Processor (SPIP) software (Image Metrology, Denmark). (41) Hutter, J.; Bechhoefer, J. ReV. Sci. Instrum. 1993, 64 (7), 1868-1873.
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Molecular Dynamics Simulation Methods. The atomic model derived from X-ray crystallography of scallop muscle myosin subfragment-242 was used as a starting point for the molecular dynamics simulations. The leucine zipper residues were removed from the atomic model, and hydrogens were added to the remaining intact residues (843-885) of the myosin subfragment-2 coiled coil. The molecule was oriented with the center of the coiled coil along the x-axis and then placed into a 228 Å × 25 Å × 33 Å TIP3 water box with four sodium ions to neutralize the system. The structure was manipulated and visualized with VMD.43 Energy minimization and molecular dynamics were performed using NAMD.44 This hydrated system was then minimized for 60 000 steps at 0 K and then equilibrated to 300 K over 20 000 steps (40 ps). The system was then subjected to 1.5 ns of molecular dynamics. The relaxed structure at the 1.5 ns simulation was used in subsequent steered molecular dynamics simulations (SMD). SMD was performed using NAMD with a spring constant of 10 kcal/(mol Å2) (6.9 N/m) and a velocity of 0.0001 Å/step (0.05 Å/ps). The simulation was run for 3000 ps for an extension of 15 nm.
Results and Discussion Force-distance spectra are operationally defined by the cantilever movement at the surface, reflecting mainly the stretching of the protein segment between the tip and substrate. The force-distance curves are subsequently interpreted, e.g., in the context of the wormlike chain model45 or the freely jointed chain model15 to obtain nanomechanical information. Typical force-distance spectra from AFM, however, are frequently composite curves consisting of contributions from both tip-surface and the tip-target interaction (Figure 2A). Figure 2B shows the occurrence of tip-surface adhesion of a Si3N4 tip interacting with a gold surface. The attractive force bends the cantilever and delays its release from the surface, causing the tip-surface spikes indicated on this figure. Typically such spikes can linger up to tens of nanometers long, obscuring valuable nanomechanical data as the cantilever retracts from the surface. To illustrate the type of nanomechanical information at low extensions, the mechanical unfolding of a 42-amino acid S2 fragment of myosin coiled-coil of R-helices was simulated by the SMD method. Parts A and B of Figure 3 show the force spectrum from the SMD simulation in water, with low-pass filtering to remove the thermal noise that obscures the larger motions of the molecular system. The principal characteristics are a rapid rise to a plateau which varies in force as the turns of the helices unfold and then a rapid rise to higher forces as the unfolded polypeptide becomes increasingly stretched. The 42residue polypeptide chains are almost completely unfolded by 8 nm of extension where the force begins to rise rapidly. As can be seen in the simulation of the short coiled-coil (Figure 3B) (and experimentally for longer coiled-coils37), there is significant variation in the plateau force as the coiled-coils unfold. This variation starts shortly after the plateau force is reached, which is less than 1 nm of extension in this simulation. This simulation demonstrates the fact that there may be significant structural information in the first few nanometers of a force spectrum that can often be obscured by tip-surface interactions. This is especially true for structures such as the coiled-coil that has structural changes at low levels of force. It (42) Li, Y.; Brown, J. H.; Reshetnikova, L.; Blazsek, A.; Farkas, L.; Nyitray, L.; Cohen, C. Nature 2003, 424 (6946), 341-345. (43) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14 (1), 33. (44) Kale, L.; Skeel, R.; Bhandarkar, M.; Brunner, R.; Gursoy, A.; Krawetz, N.; Phillips, J.; Shinozaki, A.; Varadarajan, K.; Schulten, K. J. Comput. Phys. 1999, 151 (1), 283-312. (45) Bustamante, C.; Marko, J.; Siggia, E. Science 1994, 265, 1599-1600.
Figure 2. Reduction of tip-surface sticking on functionalized OEG SAM. Typical AFM force-distance curves (right panels) and the cantilever deflections (left panels) are shown. (A) Stretching of a protein molecule myosin on the nonpassivated gold surface. (B) Tip-surface sticking caused by nonspecific adhesion forces. Convex deflection of the cantilever is caused by the tip sticking to the gold surface as it retracts. (C) No tip-surface sticking for a passivating OEG SAM on the surface. Tip-target traces are seen only when a target protein recognition event occurs. (D) Histogram analysis of pull-off forces from stretching ND66 nebulin fragments on either naked gold surface or on a mixed SAM/gold surface. A substantial portion of the pull-off forces on naked gold derived from the tip-surface interaction. In contrast, no tip-surface force was observed on SAM and the pull-off forces arose from stretching ND66 molecules. Clean gold surfaces show a marked increase in tip-surface sticking even when a low concentration of surface protein is present.
is therefore of paramount importance that such tip-surface interactions be minimized or eliminated in order to extract complete data from the low force and short extension regimes of the force curves. To illustrate this point, myosin subfragment S2 was immobilized on the mixed SAM platform as described above and the force-distance curve was obtained by stretching this protein via AFM (Figure 3C). As described in an earlier work from our group, the curves show the characteristic rise, plateau, and exponential phases of stretching an R-helical coiled-coil, before detaching from the AFM cantilever. The rise length of around 5 nm and the plateau force of 70-80 pN correspond well to the reported values for S2.37 In particular, the striking similarity
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Figure 3. Nanomechanical events at low force and small extension. (A, B) Steered molecular dynamics of stretching scallop myosin S2 coiled-coil reveals significant structural (A) and mechanical events (B) at low force-low extension regions of force-distance curves. Stretching speed is 5 m/s. (C) AFM stretching of rabbit myosin S2 R-helical coiled-coil on the SAM platform shows a striking similarity to computationally determined events. Stretching speed is 0.5 µm/s. A characteristic triphasic extension behavior with rise (R), plateau (P), and exponential (E) phases is observed (see ref 37 for details).
between the experimentally obtained curves and those obtained computationally illustrates the power of integrating the current platform with molecular modeling to gain deeper nanomechanical insights over a broad range of force and extension. The ability to resolve force-extension behavior of the S2 subfragment revealed that this flexible region of the myosin motor is able to undergo a force-induced unfolding during the acto-myosin interaction. The principal difference between the simulated and experimental spectra of coiled-coils is in the force level of the plateau. In the simulation data shown in Figure 3, the plateau force is ∼1 nN, which is about 10-fold higher than that reached experimentally. This is due, in part, to the high velocities necessary for SMD simulations to be computationally efficient. (Reduction of the velocity by a factor of 5 reduces the plateau force 2-fold.) For the experiment, the stretching speed was ∼0.5 µm/s, which is 6 orders of magnitude less than the simulation. Elimination of Tip-Surface Adhesion: OEG SAM on Ultraflat Gold. Mica,46 glass,47 and gold17 have been typical
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substrates for biological applications involving force microscopy. While mica presents an atomically flat surface, the difficulty of surface modification limits its usage for single molecule work. In addition, mica surfaces are also prone to wear at the atomic level.48 Glass is rough at an atomic level, and discrimination of nanometer-size features poses problems for imaging single molecules. Gold surfaces are relatively inert and may be easily functionalized with thiol molecules having tailorable endgroups. Epitaxially deposited gold, however, is limited to flat terraces only a few hundreds of nanometers across. Ultraflat gold prepared by template stripping39 has been suggested as an attractive alternative for preparing large-area flat surfaces. Such surfaces, with very low RMS roughness (
