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Hydrophobic and Hofmeister Effects on the Adhesion of Spider Silk Proteins onto Solid Substrates: An AFM-Based Single-Molecule Study† Michael Geisler,‡ Tobias Pirzer,‡ Christian Ackerschott,§ Simon Lud,| Jose Garrido,| Thomas Scheibel,§,⊥ and Thorsten Hugel*,‡,⊥ IMETUM, Physics Department, and CeNS, Chemistry Department, Walter Schottky Institute, and Munich Center for Integrated Protein Science, Technische UniVersita¨t Mu¨nchen, 85748 Garching, Germany ReceiVed August 1, 2007. In Final Form: October 2, 2007 AFM-based single-molecule force spectroscopy has been used to study the effect of Hofmeister salts and protein hydrophobicity on the adhesion of recombinant spider silk proteins onto solid substrates. Therefore, a molecular probe consisting of a spider silk protein and an AFM tip has been developed, which (i) is a well-defined, small system that can be simulated by molecular dynamics simulations, (ii) allows access to the whole soluble concentration range for ions, and (iii) provides the distribution of desorption forces rather than just ensemble-averaged mean values. The measured desorption forces follow the Hofmeister series for anions (H2PO4-, Cl-, I-) with a stabilizing energy of more than 15 kBT for 5 M NaH2PO4. Moreover, this effect is influenced by the hydrophobicity of the spider silk protein, indicating that hydrophobic and Hofmeister effects are closely related.
Introduction The adhesion of polymers onto solid substrates is becoming more and more important in modern technologies, especially in the creation of new composite materials, resins, and sensor devices. In most composite materials, proper adhesion strength in between the composites is crucial for the functionality of the compound. For example. the adhesion of varnish has to be extremely strong, whereas the adhesion of active agents onto drug eluting implants should be reversible and the adsorption of proteins onto the walls of biomedical devices for in vitro diagnostic systems should not take place at all. In addition, most materials such as glues should keep their functionality in aqueous environment or in medical technology should even work under physiological conditions, which underlines the importance of the influence of water and salt. Until now, it has not been possible to calculate or predict the adhesion of a compound of materials because of the complex interplay of interactions. Therefore, we have to rely on destructive testing methods for the adhesion of polymer coatings.1-3 For almost a decade, single-molecule AFM has been used to investigate the equilibrium adhesion of polymers onto solid substrates and to extract surface bonding energies.4 The equilibrium forces are characterized by plateaus in the force-extension trace and by very well defined plateau forces.5 Meanwhile, the
electrostatic contribution to the plateau force could be separated and is well known6 whereas hydrophobic interactions are far from being understood7-10 and Hofmeister effects have not been investigated at all. The main challenges with single-molecule experiments on hydrophobic substrates are the unspecific interaction between the AFM tip and substrate, aggregation of the hydrophobic polymers, and adsorption of contaminants present in air or dispersed in solution (most contaminants will quickly settle on the water-substrate interface). Finally, air bubbles are important because they considerably influence the long-range hydrophobicity.11 Despite these challenging problems, the need for detailed single-molecule investigations that are comparable to theory justifies the effort because the hydrophobic effect manifests itself in many fields such as micelle formation, protein aggregation, protein solubility, surface adhesion, and protein folding. (For recent reviews, see refs 12 and 13.) In fact, protein folding has become a major motivation for the development of a profound understanding of the hydrophobic interactions on the molecular length scale. Closely related is the Hofmeister effect that is apparent in several dozen different measurements,14 which originally described striking systematic effects of different neutral salts on the solubility of proteins. The most common are the salting in or out of proteins and the folding and unfolding of proteins.15-17
†
Part of the Molecular and Surface Forces special issue. * Author to whom correspondence should be addressed. E-mail: hugel@ imetum.tum.de. ‡ Institute of Medical Engineering, Physics Department, and CeNS. § Chemistry Department. | Walter Schottky Institute. ⊥ Munich Center for Integrated Protein Science. (1) Pocius, A. V. Adhesion and AdhesiVes Technology; Hanser Verlag: Mu¨nchen, Germany, 2002. (2) Browning, R. L.; Lim, G. T.; Moyse, A.; Sue, H. J.; Chen, H.; Earls, J. D. Surf. Coat. Technol. 2006, 201, 2970-2976. (3) Chalker, P. R.; Bull, S. J.; Rickerby, D. S. Mater. Sci. Eng. A 1991, 140, 583-592. (4) Chatellier, X.; Senden, T. J.; Joanny, J. F.; di Meglio, J. M. Europhys. Lett. 1998, 41, 303-308. (5) Hugel, T.; Grosholz, M.; Clausen-Schaumann, H.; Pfau, A.; Gaub, H.; Seitz, M. Macromolecules 2001, 34, 1039-1047.
(6) Seitz, M.; Friedsam, C.; Jostl, W.; Hugel, T.; Gaub, H. E. ChemPhysChem 2003, 4, 986-990. (7) Friedsam, C.; Gaub, H. E.; Netz, R. R. Europhys. Lett. 2005, 72, 844-850. (8) Cui, S. X.; Liu, C. J.; Wang, Z. Q.; Zhang, X. Macromolecules 2004, 37, 946-953. (9) Haupt, B. J.; Senden, T. J.; Sevick, E. M. Langmuir 2002, 18, 2174-2182. (10) Shi, W. Q.; Wang, Z. Q.; Cui, S. X.; Zhang, X.; Bo, Z. S. Macromolecules 2005, 38, 861-866. (11) Meagher, L.; Craig, V. S. J. Langmuir 1994, 10, 2736-2742. (12) Chandler, D. Nature 2005, 437, 640-647. (13) Meyer, E. E.; Rosenberg, K. J.; Israelachvili, J. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15739-15746. (14) Collins, K. D.; Washabaugh, M. W. Q. ReV. Biophys. 1985, 18, 323-422. (15) Hofmeister, F. Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol. (Leipzig) 1888, 24, 247. (16) Tadeo, X.; Pons, M.; Millet, O. Biochemistry 2007, 46, 917-923. (17) Vogel, R. Curr. Opin. Colloid Interface Sci. 2004, 9, 133-138.
10.1021/la702341j CCC: $40.75 © 2008 American Chemical Society Published on Web 11/28/2007
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The origin of these observations is still a vital issue12,18-20 and is probably closely related to the hydrophobic interaction, which is also under discussion.12,13,21 To gain a basic understanding of these interactions at the molecular level, we developed an AFM-based molecular probe that is capable of measuring interactions between well-defined spider silk proteins with different hydrophobicities and hydrophobic substrates in a variety of salt solutions. This allows us to track the influence of various parameters on the interaction between single polymers and solid substrates and to extract surface binding energies. In addition, because of its small size and proximity to equilibrium, this system can be treated with molecular dynamics (MD) simulations.22 The MD simulations allow us to extract all of the different contributions to the adhesion free energy (see below) and provide completely new insight into interactions at the molecular level. In this study, we focus on the contribution of salts and polymer hydrophobicity on single polymer surface binding energies and the interplay between hydrophobic and Hofmeister effects. Materials and Methods AFM. For the AFM measurements in this study, we used a MFP-3D (Asylum Research, Santa Barbara, CA) and silicon nitride cantilevers (MLCT-AUHW from Veeco GmbH, Mannheim, Germany). Diamond Surfaces. The diamond substrates used in this work are polished (root-mean-square roughness of less than 0.3 nm, measured over an area of 5 µm × 5 µm) polycrystalline diamond films (ElementSix Advancing Diamond Ltd., U.K.) of size 5 mm × 5 mm × 0.5 mm. Hydrophobic diamond surfaces are obtained after hydrogen termination of the surface.23 Hydrogenation is performed in a vacuum chamber using a hydrogen gas flow of 300 sccm over a hot (2000 °C) tungsten filament, placed at a distance of 4 cm from the substrate. During the process, the sample temperature (approximately 700 °C) is determined with a thermocouple. Static contact angle experiments, using a 0.2 µL deionized water droplet placed on top of the diamond substrate, are carried out for the characterization of the surface hydrophobicity. All values are obtained from at least six measurements. The derived water contact angle for a freshly prepared hydrogen-terminated diamond surface is 90 ( 1°, so the surface is rather hydrophobic. Production of Recombinant Proteins C16 and (QAQ)8NR3. Gene constructs for C16 and (QAQ)8NR3 were cloned as described previously.24,25 (QAQ)8NR3 comprises the A and Q motifs derived from ADF-3 (Araneus diadematus fibroin), and C16 comprises the C motif derived from ADF-4. All sequences are derived from the repetitive core domain of the respective spider silk protein. The motifs are synthetically adapted to the codon usage of Escherichia coli. Each construct was finally cloned into the pET21a expression vector (Novagen). E. coli BLR(DE3) (Novagen) cells were transformed with the resulting plasmids and grown in LB medium with 100 µg/mL ampicillin at 37 °C until OD600 ) 0.5 was reached.26 Cells were shifted to 30 °C for (QAQ)8NR3 and to 25 °C for C16 before induction with 1 mM isopropyl β-D-galactopyranoside (IPTG) for 3 h. E. coli (18) Baldwin, R. L. Biophys. J. 1996, 71, 2056-2063. (19) Kunz, W.; Lo Nostro, P.; Ninham, B. W. Curr. Opin. Colloid Interface Sci. 2004, 9, 1-18. (20) Cacace, M. G.; Landau, E. M.; Ramsden, J. J. Q. ReV. Biophys. 1997, 30, 241-277. (21) Urry, D. W. Chem. Phys. Lett. 2004, 399, 177-183. (22) Horinek, D.; Serr, A.; Geisler, M.; Pirzer, T.; Garrido, J.; Scheibel, T.; Hugel, T.; Netz, R. R. submitted to Proc. Natl. Acad. Sci. U.S.A., 2007. (23) Hartl, A.; Garrido, J. A.; Nowy, S.; Zimmermann, R.; Werner, C.; Horinek, D.; Netz, R.; Stutzmann, M. J. Am. Chem. Soc. 2007, 129, 1287-1292. (24) Huemmerich, D.; Helsen, C. W.; Quedzuweit, S.; Oschmann, J.; Rudolph, R.; Scheibel, T. Biochemistry 2004, 43, 13604-13612. (25) Vendrely, C.; Scheibel, T. Macromol. Biosci. 2007, 7, 401-409. (26) Schmidt, M.; Ro¨mer, L.; Strehle, M.; Scheibel, T. Biotechnol. Lett. 2007, 29 (11), (DOI10.1007/s10529-007-9461-z).
Figure 1. Principle of the desorption experiment. C16 is covalently attached to an AFM tip (top) and then brought into contact with a solid substrate (bottom). Finally, the force during retraction of the AFM tip and therefore desorption of the C16 is measured. For covalent attachment, NHS active ester reacts with the N terminus to form a peptide bond. R1 represents the PEG spacer, and R2 replaces the spider silk amino acid sequence. cells were harvested after induction for 3 to 4 h, and the cell pellets were resuspended in 20 mM HEPES, 100 mM NaCl at pH 7.5 (5 mL/g of cells). Upon addition of 0.2 mg of lysozyme (Sigma-Aldrich, St. Louis, MO) per mL, the resuspension was incubated at 4 °C for 30 min until becoming viscous. Protease inhibitor (Serva, Heidelberg, Germany) was added before the cells were ultrasonicated using an HD/UW2200/KE76 ultrasonicator (Bandelin, Berlin, Germany). Contaminating DNA was digested upon addition of 10 µg/mL DNase and 3 mM MgCl2 (protease-free DNAse was obtained from Roche, Mannheim, Germany) and incubation at 4 °C for 30 min. Upon sedimentation at 50 000g and 4 °C for 30 min, remaining soluble proteins were incubated at 80 °C for 20 min. A further centrifugation step was performed, and C16 or (QAQ)8NR3 was found in the supernatant. Upon addition of 20% ammonium sulfate the recombinant spider silk proteins precipitated. (QAQ)8NR3 pellets were rinsed with the ammonium sulfate, whereas C16 pellets were washed with 8 M urea. C16 and (QAQ)8NR3 were resuspended in 6 M guanidinium thiocyanante and dialyzed against 10 mM ammonium hydrogencarbonate at 4 °C. Finally, proteins were lyophilized and stored at -20 °C until use. Tip Preparation. Chemical as well as plasma treatments were used to clean the AFM cantilevers and increase the number of surface reactive groups to ensure dense coverage with amino groups. Aminofunctionalized tip surfaces for the covalent coupling of spider silk protein molecules were obtained by using Vectabond Reagent (Axxora, Lo¨rrach, Germany) on the activated silicon nitride tips. C16 spider silk was attached by keeping the aminated cantilevers for half an hour in an aqueous polyethylene glycol solution (1:500 mixture of the 6 kg mol-1 PEG-R,ω-Di-NHS ester heterobifuntional linker and the 5 kg mol-1 monofuntional CH3O-PEG-NHS ester, RappPolymere, Tu¨bingen, Germany) and afterward in a C16 solution (approximately 0.5 mg mL-1) for another 2.5 h. PEG was used as spacer and inert back-filling molecule to passivate the tip surface and isolate the contribution of the spider silk from the unspecific adhesion to the tip. A stable amide bond is formed by the reaction of the N-hydroxysuccinimidyl (NHS) active ester with the N-terminus of the spider silk as shown in Figure 1. Force Spectroscopy and Analysis. All force spectroscopy experiments with C16 were performed at room temperature in a closed fluid cell filled with a 0.5 or 5 M solution of the salt under investigation, and the experiments on (QAQ)8NR3 were performed
1352 Langmuir, Vol. 24, No. 4, 2008 in a 40 µL salt solution droplet at a concentration of 4 M. The functionalized tip with the spider silk protein is brought into contact with the solid substrate for 1 s to adsorb and then retracted at a constant speed of 1 µm s-1. Force-extension traces were obtained from the deflection piezopath signal as described elsewhere.27 They show no dependency on the pulling velocity in the typical range for single-molecule experiments with an AFM of 0.1 to 10 µm s-1. In addition, the plateau heights do not change upon degassing of the buffer. The traces were taken at least at two different positions on the hydrogen-terminated diamond. In AFM experiments, there is always a slight variation in the inverse optical lever sensitivity (InvOLS) due to optical lever fluctuations, local solvent refraction index changes, or changes in the cantilever-surface interaction during indentation. To minimize this error, we averaged the InvOLS of the first five as well as the last five traces and used it for the whole experiment until either the salt solution or the cantilever was changed. The spring constant of each cantilever was determined prior to each experiment. We determined the spring constant by integrating over the power spectrum of the MFP (which is already corrected for the κ factor) from 75 Hz to the first minimum and using the equipartition theorem28 k〈∆x〉2 ) kBT. The errors for the desorption forces are determined as follows. First, the standard deviation for a single measurement is calculated, and then the rms (root-mean square) of all of the standard deviations is determined to give the total statistical error. The calibration error (uncertainties in spring constant and inverse optical lever sensitivity) is discussed at the end of the Molecular Adhesion Probe section. Contact angle measurements were made in a home-built goniometer equipped with a CCD camera. For the determination of the angles, we used Java-based freeware ImageJ with the drop analysis plugin.29 Both angles of the drop image were recorded at three different positions on the surface for each salt solution, and the mean value was calculated. AFM Imaging. A (10 µm × 10 µm)-sized surface area of the hydrophobic diamond was investigated by contact mode AFM with a scan rate of 0.6 Hz at a resolution of 512 × 512 data points. Suitable cantilevers were chosen for imaging (OMCL-RC800PSA, Olympus, Japan).
Results and Discussion Diamond Surface. The Diamond surface was stored in DMSO and rinsed with water, acetone, and water before each measurement. Figure 2 shows an image of the diamond surface after many measurements. The peak-to-peak elevation is still less than 1 nm over a distance of 10 µm, and the surface is still very clean. Therefore, the contribution from surface roughness or contamination is negligible and will not be discussed further. The contact angle of water decreased slightly to about 80° (cf. Table 1). Molecular Adhesion Probe. In the following text, we call the combination of AFM tip and single spider silk protein the molecular (adhesion) probe. Before describing the influence of various salts and polymer hydrophobicitiy on the adhesion properties, we give a thorough characterization of our molecular probes. As depicted in Figure 1 and described in the Materials and Methods section, single spider silk proteins are covalently attached to an AFM tip via their N terminus to form the molecular probe. The strong interaction between the tip and the hydrophobic surface may mask the desorption event of a single probe molecule. In the present study, unspecific interactions between the AFM tip and the underlying substrate are suppressed by passivation of the tip with PEG. Figure 3 shows the effect of PEG spacers differing in molecular weight on the suppression of this unspecific adhesion. Clearly, PEG with a molecular weight of 6 or 10 kg (27) Hugel, T.; Seitz, M. Macromol. Rapid Commun. 2001, 22, 989-1016. (28) Butt, H. J.; Jaschke, M. Nanotechnology 1995, 6, 1-7. (29) Stalder, A. F.; Kulik, G.; Sage, D.; Barbieri, L.; Hoffmann, P. Colloids Surf., A 2006, 286, 92-103.
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Figure 2. AFM image of a 10 µm × 10 µm surface area of the hydrogen-terminated diamond surface and a corresponding cross section (blue line). Table 1. Summary of the Main Results desorption forces (pN) salt (5 M)
C16
QAQ8 NR3
contact angle (deg)
NaI NaCl NaH2PO4
(106 ( 19) (126 ( 16) (280 ( 44)
(71 ( 11) (99 ( 12) (124 ( 16)
(79 ( 2) (85 ( 2) (89 ( 3)
mol-1 can significantly reduce the adhesion peak. In addition, by the use of spacers the probe molecule shifts away from the tip apex, increasing the separation at which the force drops to zero (rupture length). Furthermore, the sharpness of the distribution increases with spacer length, likely attributed to the passivating properties of the underlying PEG layer. The defined rupture length is henceforth used to identify the spider silk proteins unambiguously. In Figure 4, a couple of force-extension traces for the desorption of the polymers on different days and for different cantilevers are shown with the corresponding desorption force distributions. The solid substrate is an H-terminated diamond surface with a contact angle of about 80°, and the spider silk protein is in this case the C16 motive. We used 6 kg/mol PEG because we got a much better yield of single molecules attached to the AFM tip than with the 10 kg/mol PEG. The distribution of rupture length is always similar to the one shown in Figure 3 for the 6 kg/mol PEG. In more than 65% of the experiments, we have a mean of about 230 nm, which is in good agreement with the estimated lengths (as follows). We conclude that in general we managed to tether the protein covalently at its N-terminal end. Shorter plateaus might result from the strong unspecific adhesion of part of the chain to the cantilever or from attachment at one of the 18 glutamines contained in C16. The expected length can be estimated from the extended contour length: With 575 amino acids from the C16 spider silk (0.365 nm each) and 136 CH2CH2O monomers (0.36 nm/monomer in the all-trans conformation, which is almost exclusively occupied at a force of 60 pN), we get about 260 nm total. Considering an extension of about 85% at a force of 60 pN,30 we expect about (30) Neuert, G.; Hugel, T.; Netz, R. R.; Gaub, H. E. Macromolecules 2006, 39, 789-797.
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Figure 3. Four different desorption experiments with C16 in 20 mM phosphate buffer. The cantilever is brought into contact with the surface (red trace). Then the tip is retracted, and the desorption force and rupture length of the molecule are measured (black trace). We used different molar masses of PEG ranging from 2 to 10 kg/ mol. The upper force-extension trace represents measurements with the protein directly coupled to the functionalized tip without spacers. As depicted by the corresponding length histograms in the right panel, the use of longer spacers not only causes the rupture length to increase but also results in a sharper rupture length distribution. Traces are vertically offset by 1 nN.
220 nm. This is in good agreement with the observed values considering the 1.13 polydispersity of the PEG linker. The defined length of the protein reflects a good fingerprint of the investigated polymer and allows an unambiguous characterization of the acting forces. Upon restriction to plateaus with the expected rupture length (50%, very small force distributions with an error of less than 6% are obtained (Figure 4). With one C16 molecule covalently bound on average to the bottom of the cantilever tip, we recorded up to 2000 force-extension traces with the same molecule in various buffers. The long-term stability of the probe reached by covalent bonds allowed a direct comparison of the desorption forces at different buffer conditions. If we compare experiments from different days with different cantilevers, the deviation between experiments is slightly higher than the error from the force distributions because of the uncertainties in the determination of the spring constant and InvOLS. To estimate this error, we performed one experiment seven times with different cantilevers. Figure 5 shows the result of these experiment and allows us to estimate the standard deviation to about 8%. Each experiment in this paper was reproduced at least once on a different day with a different cantilever chip. Hofmeister Effect. Next we compared the effect of various salts on the desorption forces. We chose concentrations of 0.5 and 5 M because the Hofmeister effect increases with salt concentration and all possible charge effects are screened and the salts are still not crystallizing. Figure 6 shows the measured
Figure 4. Five out of seven different experiments performed in 0.5 M NaCl solution with a 6 kg mol-1 PEG spacer. Each is represented by a typical force-distance trace and the corresponding force distribution. The deviation of the mean values is shown in Figure 5. Traces are vertically offset by 2 nN.
Figure 5. Variation of the desorption forces from seven experiments with different cantilevers performed on different days. (See also Figure 4.) The dashed line marks the force average.
desorption force taken with the single spider silk protein attached to an AFM tip as described before. The comparison of the forces acting at different salt concentrations of our solutes demonstrates that Hofmeister effects are either not present or very weak at medium salt concentration, but are very strong in the high-salt regime. As expected, phosphate strongly increases the desorption force, and chloride has a more
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Figure 6. Desorption force of C16 on diamond in NaCl, NaI, and NaH2PO4 solutions. At a concentration of 0.5 M, there is no measurable influence of the ions (black bars), whereas a clear impact of the anions becomes visible at 5 M (red bars). The trend follows the Hofmeister series.
Figure 7. Desorption forces (O) for C16 from H-terminated diamond, the change in ProtL mid-denaturing temperatures, ∆T, upon the addition of 0.8 M salt16 (2), and the relative solubility of benzene in 1 M salt32 (0). Arrows assign the symbols to the corresponding scale.
stabilizing effect than iodide. This effect is in agreement with the stabilizing power of anions in salting out proteins, as reported in hundreds of publications (ref 14 and references therein):31 H2PO4- > ... > Cl- > NO3- > I-. The more stabilizing salts (H2PO4-) are generally called kosmotropic; they promote the salting out and folding of proteins. In Figure 7, we compare our results with studies on the effect of Hofmeister ions on protein solubility32 and protein denaturation16 corresponding to observations made in solution. As mentioned before, the general trend at high salt concentration is the same. However, the graph demonstrates that the stabilizing effect is much more pronounced in our single-molecule experiments. This could be due to the very high salt concentration (which cannot be accessed by bulk experiments) or the large hydrophobic surface. Neglecting a small entropic conformational contribution,7 the average adsorption free energy per amino acid can be estimated as W ) 0.365 nm × force.33 This is abaut 9kBT, 11kBT, and 25kBT for NaI, NaCl, and NaH2PO4, respectively (kBT ) 4.2 pN nm at a temperature of 300 K). The stabilizing energy (the additional adsorption free energy upon the addition of salt) for the 5 M phosphate buffer is therefore more than 15kBT per amino acid. This is a huge energy gain, compared to typical equilibrium energies for protein folding or the unzipping of coiled coils with an energy of about 0.5kBT per amino acid.34 Water-water interaction has an energy of up to 10kBT at room temperature.35 (31) Zoldak, G.; Sprinzl, M.; Sedlak, E. Eur. J. Biochem. 2004, 271, 48-57. (32) Mcdevit, W. F.; Long, F. A. J. Am. Chem. Soc. 1952, 74, 1773-1777. (33) Hanke, F.; Livadaru, L.; Kreuzer, H. J. Europhys. Lett. 2005, 69, 242248.
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Figure 8. Desorption forces of hydrophobic C16 on diamond in the electrolytes discussed above (red) are shown together with desorption forces of less-hydrophobic (QAQ)8NR3 (blue).
Figure 9. Contact angles of the 5 M salt solutions measured on the hydrophobic diamond (thin black bars in front). They follow the same trend, but not the same function, as molecular desorption forces of C16 (broad red bars behind).
In summary, the adsorption free energy of a (mildly) hydrophobic polymer on a hydrophobic substrate follows a Hofmeister salt series for very high salt concentrations, but the behavior differs from the protein-protein interaction free energy, where the Hofmeister salt effect is already pronounced at 0.5 M. This might be due to the nearly infinite dilution of the peptide at the tip. Hydrophobic Effect. Finally, we checked this series on a second engineered spider silk protein (QAQ)8NR3, revealing a similar general trend (Figure 8). What is the effect of the polymer’s hydrophobicity on the desorption force? (QAQ)8NR3 is less hydrophobic than C16,24,25 namely, -0.46 for C16 and -0.92 for (QAQ)8NR3 (calculated from Kyte et al.36), leading to stronger adhesion and therefore a higher desorption force for C16 on a hydrophobic substrate (all charges should be screened), as shown in Figure 8. Furthermore, the stabilizing power of Hofmeister ions is much weaker on the less hydrophobic (QAQ)8NR3 (15kBT for C16 compared to 5kBT for (QAQ)8NR3), indicating a strong relation between Hofmeister effects and hydrophobic effects. Contact Angles. For completeness, the contact angles θ for the used salt solutions are shown in Figure 9. They are related to the adhesion energy per unit area, ∆WSLV, of the solid and liquid surfaces in medium V and the surface tension of the liquid, γ, by the Young-Dupre´ equation:
γ(1 + cos θ) ) ∆WSLV To first approximation, for angles close to 90°, ∆WSLV and θ are proportional, and the change in contact angle should (34) Bornschlogl, T.; Rief, M. Phys. ReV. Lett. 2006, 96, 118102. (35) Finney, J. L. Philos. Trans. R. Soc. London, Ser. B 2004, 359, 11451163. (36) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105-132.
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therefore directly represent the change in adhesion energy. Changes in contact angle follow the same order as polymer adhesion but definitely not the same function, indicating again that there is “no ONE factor involved in Hofmeister effects”.19
Concluding Remarks Are the presented results just a new manifestation of the Hofmeister effect? In principle yes, but this system offers a completely new quality of data that cannot be obtained by any other method: (i) the small size of the system allows a direct comparison with MD simulations, (ii) the quasi-infinite dilution of polymer allows access to the whole soluble concentration range for ions, (iii) distributions of physical values are obtained, and different populations are therefore not hidden in average values, and (iv) the effect of salt and polymer hydrophobicity can be investigated and separated. In addition, we overcome all limitations that made force measurements across liquids by AFM “an art form”.19 For these reasons, such experiments are expected to lead to a better understanding of Hofmeister and hydrophobic effects. Of course, more measurements at many different salt concentrations and with various ions and polymers are necessary to put existing theoretical models to the test and to allow for new (unified) descriptions. One could try to interpret the results of this study in terms of a model based on existing theories, but no theory to date accounts for all contributions to describe the interaction free energy of water, ions, and polymers close to a solid surface. For our
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experiments, we would have to consider the changes upon desorption in the following contributions: water-water, waterpolymer, water-surface, polymer-surface, polymer-polymer, water-ions, surface-ions, and polymer-ions. Some of them might then be named hydrophobic and others might be related to Hofmeister effects, but differentiation could become meaningless in the case of polymer desorption from solid substrates. MD simulations by Horinek et al.37 point out that all water structure and dispersive contributions could become relevant and dominant depending on the system under investigation. Because most of the contributions in our system listed above are separately much larger than their sum, neglecting some of the contributions might give the correct free-energy change for the presented data, but the restriction to some contributions will then very likely fail to describe other systems. Only a complete description including all impacts will provide us deeper insights. Acknowledgment. We thank the Stiftung Industrieforschung, the DFG, and the Fond der Chemischen Industrie for financial support. M.G. is supported by the Elitenetzwerk Bayern. Helpful discussions with Matthias Rief, Roland Netz, and Dominik Horinek are gratefully acknowledged. LA702341J (37) Horinek, D.; Serr, A.; Bonthuis, D.; Bostro¨m, M.; Kunz, W.; Netz, R. R. Molecular Hydrophobic Attraction and Ion-Specific Effects Studied by Molecular Dynamics. Langmuir, in press, 2007.
