pubs.acs.org/Langmuir © 2010 American Chemical Society
Mechanisms of Protein Adhesion on Surface Films of Hydrophobin Zefang Wang,†,‡ Michael Lienemann,† Mingqiang Qiau,‡ and Markus B. Linder*,† †
VTT Biotechnology, Technical Research Centre of Finland, Tietotie 2, FIN-02044VTT, Espoo, Finland, and ‡ The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, No. 94 Weijin Road, Tianjin 300071, P. R. China Received November 11, 2009. Revised Manuscript Received April 23, 2010
Hydrophobins are adhesive proteins produced by filamentous fungi. They are in many cases secreted into the medium and adsorb readily to a number of different surfaces. They fulfill many different tasks such as the formation of various coatings and mediating adhesion of fungi to surfaces. The mechanism of how hydrophobins adhere and how they mediate fungal adhesion is of interest both from the point of view of fungal biology and for various biotechnical immobilization applications. It has been shown that hydrophobins typically form a monomolecular layer on solid substrates. We are especially interested in how a surface layer of hydrophobin can mediate the adhesion of a second layer of another protein. In this work we systematically studied how proteins adsorb onto hydrophobins that are bound as monomolecular layers on nonpolar surfaces. We found that several types of proteins readily adsorb onto hydrophobins, but only under defined conditions of pH and ionic strength. The binding conditions were also highly dependent on the adhering protein. By studying solution conditions such as pH and ionic strength, we conclude that the surface adhesion is due to selective Coulombic charge interactions. We conclude that hydrophobins can transform a nonpolar surface into one that efficiently recruits other proteins by charge interactions.
1. Introduction In this work we have investigated the basic mechanisms of how films of adhesive proteins called hydrophobins function as anchoring layers for immobilizing a second layer of a different protein. Formation of such layered structures is likely to be a natural function of hydrophobins and can be useful also for protein immobilization in for example biosensor applications. Hydrophobins are surface active proteins produced by filamentous fungi. They have diverse roles in fungal growth and development, with functions ranging from coating of spores to adhesion to surfaces and forming structures in cell walls and fruiting bodies. They can be highly expressed and are often secreted by fungi into the environment.1-5 On the basis of sequence comparisons, hydrophobins can be divided into two main groups, class I and class II. Functionally the two classes differ in that the class I members typically form assemblies and aggregates that are much more difficult to dissolve than assemblies of class II members. One of the characteristics of class I members is that they form the amyloid-like structures that are called rodlets. Hydrophobins typically show a large variation in primary sequence. Even within each group there can be a large diversity in sequences. However, it seems that hydrophobins always share the same pattern of eight Cys residues in their sequences, having the second and third and the sixth and seventh always occurring as pairs following each other directly in sequence. The molecular structures of a few hydrophobins have been solved, showing that their structures have the basic characteristics *Corresponding author. (1) Linder, M. B.; Szilvay, G. R.; Nakari-Setala, T.; Penttila, M. E. FEMS Microbiol. Rev. 2005, 29(5), 877–896. (2) Scholtmeijer, K.; Wessels, J. G. H.; Woster, H. A. B. Appl. Microbiol. Biotechnol. 2001, 56(1-2), 1–8. (3) Wessels, J. G. H. Adv. Microb. Physiol. 1997, 38, 1–45. (4) Whiteford, J. R.; Spanu, P. D. Mol. Plant Pathol. 2002, 3(5), 391–400. (5) Wosten, H. A. B.; de Vocht, M. L. Biochim. Biophys. Acta 2000, 1469(2), 79–86.
Langmuir 2010, 26(11), 8491–8496
of an amphiphilic molecule.6 The protein structure shows a patch that is solely composed of aliphatic and thus hydrophobic side chains. The details of the structures differ between class I and II members.7 In class II, this patch is flat and comprises about 19% of the total surface area of the protein.8 The rest of the protein surface shows the typical hydrogen-bonding side chains found widely in hydrophilic protein surfaces. Hydrophobins have been much studied for their surface adhesion properties. In studies using class II hydrophobins it has been shown that oriented, monomolecular layers are formed on surfaces.9-11 In one report it was interestingly found that hydrophobins were able to recruit enzymes to solid surfaces and thereby speed up the degradation of a solid substrate.12 This type of adhesive behavior may be related to the reported functions of hydrophobins as aiding fungal adhesion when pathogenic fungi colonize their hosts.13 In some reports it has been suggested that hydrophobins can be used as an anchoring layer to immobilize functional proteins in for example biosensor applications. In these reports different types of surfaces have been first coated with hydrophobin, and then some other functional protein such as an enzyme or binding protein like IgG has been added. Somewhat surprisingly, it was found that this procedure resulted in a highly (6) Hakanpaa, J.; Paananen, A.; Askolin, S.; Nakari-Setala, T.; Parkkinen, T.; Penttila, M.; Linder, M. B.; Rouvinen, J. J. Biol. Chem. 2004, 279(1), 534– 539. (7) Sunde, M.; Kwan, A. H.; Templeton, M. D.; Beever, R. E.; Mackay, J. P. Micron 2008, 39(7), 773–84. (8) Hakanpaa, J.; Szilvay, G. R.; Kaljunen, H.; Maksimainen, M.; Linder, M.; Rouvinen, J. Protein Sci. 2006, 15(9), 2129–2140. (9) Linder, M.; Szilvay, G. R.; Nakari-Setala, T.; Soderlund, H.; Penttila, M. Protein Sci. 2002, 11(9), 2257–2266. (10) Paananen, A.; Vuorimaa, E.; Torkkeli, M.; Penttila, M.; Kauranen, M.; Ikkala, O.; Lemmetyinen, H.; Serimaa, R.; Linder, M. B. Biochemistry 2003, 42(18), 5253–5258. (11) Szilvay, G. R.; Paananen, A.; Laurikainen, K.; Vuorimaa, E.; Lemmetyinen, H.; Peltonen, J.; Linder, M. B. Biochemistry 2007, 46(9), 2345–2354. (12) Takahashi, T.; Maeda, H.; Yoneda, S.; Ohtaki, S.; Yamagata, Y.; Hasegawa, F.; Gomi, K.; Nakajima, T.; Abe, K. Mol. Microbiol. 2005, 57(6), 1780–1796. (13) Holder, D. J.; Keyhani, N. O. Appl. Environ. Microbiol. 2005, 71(9), 5260– 5266.
Published on Web 05/03/2010
DOI: 10.1021/la101240e
8491
Article
Wang et al.
functional surface where the second protein was immobilized efficiently and retained its biological function.14 In the work reported here we studied systematically the effects of adsorption conditions to gain an understanding of the mechanisms behind the hydrophobins role as an anchoring layer. We used two types of hydrophobin, one belonging to class I (HGFI) and one belonging to class II (HFBI). As the main analytical tool, a quartz crystal microbalance with dissipation monitoring (QCM-D) was used. The instrument measures the mass of bound materials and the viscoelastic properties of the bound layer.15 The viscoelasticity is quantified as the dissipation value (D) and is a measure of how rapidly the oscillations decay. A quick decay corresponds to a viscous surface, and a slower decay corresponds to a more rigid surface.
2. Materials and Methods Reagents and Chemicals. HGFI was purified from aerial hyphae of Grifola frondosa as described earlier,16 except that a 3 mL Resource RPC column (Pharmacia Biotech, Sweden) was used for the chromatography. HFBI was purified from Trichoderma reesei mycelium and purified by reversed phase chromatography as described earlier.17 Glucose oxidase from Aspergillus niger (GOx) and bovine serum albumin (BSA) were obtained from Sigma Co., and chicken egg avidin was a gift from professor V. Hytonen (University of Tampere, Finland). Monoclonal IgG anti-CRP (clone 6404)18 was obtained from Medix Biochemica (Finland). This antibody belongs to the IgG1 subclass. The buffers (10 mM) used in pH test were glycine-HCl (pH 2.0, 3.3), sodium acetate-acetic acid (pH 4.0, 4.2, 4.5, 4.7, 5.0, 5.5, 5.7, and 6.0), NaH2PO4-NaOH (pH 7.0), and glycineNaOH (pH 9.0, 10.0, 11.0). Sodium acetate-acetic acid (pH 5.0) and NaH2PO4-NaOH (pH 7.0) buffers were also used in salt tests at six different concentrations (1, 10, 100, 200, 300, and 500 mM). Both HFBI and HGFI were dissolved in the corresponding buffers to a concentration of 100 μg/mL. QCM Measurements. A quartz crystal microbalance was used for the simultaneous measurement of frequency and dissipation (D4-QCM system, Q-Sense AB, Sweden). The gold-coated quartz crystal sensor disks were coated with 1-hexanethiol as follows: First, sensor disks were cleaned in a standard UV/ozone chamber for 10 min and exposed to a heated H2O/NH3/H2O2 mixture (5:1:1) for another 10 min followed by thorough rinsing with Milli-Q water. Then dried sensor disks were immersed in pure ethanol for 2 min to sensitize the surfaces. The wet sensor disks were then immersed in 50 mM 1-hexanethiol/ethanol solution overnight at room temperature. Finally, they were rinsed with pure ethanol and Milli-Q water, dried with N2, and mounted in the measurement chambers. For both HFBI and HGFI, 0.1 mg/mL protein solutions (400 μL) were pumped through the measurement chambers using a flow of 100 μL/min. The sensor was incubated until the resonance frequency (third overtone) was stable (30-60 min), and the surface was then washed with the buffer to be used for studying the pH-dependent protein association. Avidin, GOx, and BSA were dissolved in the appropriate buffer to a concentration of 1 mg/mL and injected as described above. IgG was used at 100 μg/mL. (14) Qin, M.; Hou, S.; Wang, L.; Feng, X.; Wang, R.; Yang, Y.; Wang, C.; Yu, L.; Shao, B.; Qiao, M. Colloids Surf., B 2007, 60(2), 243–249. (15) Hook, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95(21), 12271–12276. (16) Yu, L.; Zhang, B. H.; Szilvay, G. R.; Sun, R.; Janis, J.; Wang, Z. F.; Feng, S. R.; Xu, H. J.; Linder, M. B.; Qiao, M. Q. Microbiology 2008, 154, 1677–1685. (17) Linder, M.; Selber, K.; Nakari-Setala, T.; Qiao, M. Q.; Kula, M. R.; Penttila, M. Biomacromolecules 2001, 2(2), 511–517. (18) Kapyaho, K.; Tanner, P.; Karkkainen, T.; Weber, T. Scand. J. Clin. Lab. Invest. 1989, 49(4), 389–393.
8492 DOI: 10.1021/la101240e
Figure 1. (A) Adsorption of the hydrophobins HFBI and HGFI on 1-hexanethiol surfaces as a function of pH. (B) The change is dissipation during binding. Circles are HFBI and squares are HGFI.
3. Results Adsorption of HFBI and HGFI on 1-Hexanethiol Surfaces. At first the binding of HFBI and HGFI to the hydrophobic 1-hexanethiol surface was measured. Binding data at 12 selected pH values are shown in Figure 1A (see below and Figure 2 for details on the measurement technique). For HFBI there is a variation in binding from 150 to 280 ng/cm2 depending on the pH, with the lowest binding at high and low pH and a maximum binding slightly below the calculated pI (pI=5.7). HGFI exhibits a similar behavior, except that it shows more variation in its binding as a function of pH. For HGFI, the maximum binding occurs at a pH slightly above its pI (pI = 3.3). The dissipation values for the adsorbed surfaces were measured simultaneously and are shown in Figure 1B. We note that the curve for HFBI is very stable and has very low dissipation values, indicating a highly rigid layer.15 HGFI, on the other hand, shows a different behavior with a peak in the dissipation change at around pH 4.5. Overall, the change in dissipation is higher than for HFBI, but the values still indicate a rigid layer at pH values above 6. Adsorption of GOx on Surface Layers of HFBI. In Figure 2A, a QCM sensogram of GOx on HFBI at pH 5.5 is shown as a representative example of the measurement of protein adhesion. The x-axis shows time and the y-axis the change of frequency of the third overtone. Binding to the surface is seen as a decrease in frequency. At the point indicated by A (3 min), HFBI was injected; at point B buffer was injected (42 min); at point C (85 min) GOx was injected, and at point D (147 min) buffer was again injected. At point E (200 min), at the end of the wash procedure Langmuir 2010, 26(11), 8491–8496
Wang et al.
Article
Figure 2. (A) A QCM sensogram of HFBI and GOx binding to a 1-hexanethiol surface at pH 5.5. The different points correspond to (A) HFBI injection, (B) buffer wash, (C) GOx injection, (D) buffer wash, and (E) end-point. (B) Plot of GOx binding to a hydrophobincoated surface at different pH values. Data points correspond to the difference between points C and E in QCM sensograms as described above but performed at different pH. The thick line below the curve (denoted pIHFBI-pIGOx) indicates the region where HFBI and GOx have opposite charges according to their pI values.
the final bound amount was recorded. We note that the initial binding of HFBI to the hydrophobic surface was very quick and that practically no HFBI was removed from the surface during rinsing. The change in dissipation (ΔD) value changed from zero at A to 0.3 10-6 at B. There was no change in ΔD from B to C. The binding of GOx to the HFBI surface (C to D) was slower than HFBI binding to the surface and showed a greater change in ΔD, from 0.3 10-6 to 1.3 10-6. Interestingly, some of the bound GOx was removed during the buffer rinsing (D to E), and the ΔD again decreased somewhat from 1.3 10-6 to 1.0 10-6. We interpret this result so that some GOx molecules were loosely bound to the HFBI layer and were removed by the shear force during rinsing. However, it should be noted that these values are small and that the ΔD values indicate rigid layers for the GOx. Figure 2B shows the binding (corresponding to the difference between points C and E) determined by recording sensograms corresponding to that shown in Figure 2A at different pH values. The change in resonance frequency has been converted to bound mass using the Sauerbrey equation.15 A high binding of GOx to the HFBI film was seen roughly between pH 4 and pH 6. Above and below these pH values the binding was negligible. In order to evaluate the variance of the data around the average GOx binding to the HFBI layer, triplicate measurements were Langmuir 2010, 26(11), 8491–8496
Figure 3. Adsorption of different proteins on HFBI and HGFI hydrophobin surfaces. Experiments correspond to those described in the caption of Figure 2. (A) Binding of BSA. (B) Binding of avidin. (C) Binding of IgG. The thick lines below the binding curves (denoted pI;pI) indicate regions where hydrophobins and adsorbing proteins have opposite charges according to their pI values.
performed at pH values where high and low binding was observed. The data were used to calculate standard errors of the measurement points. For points where high adsorption occurred (pH 4.7) the standard error was 5%. For binding points where low binding occurred (pH 6.0) the standard error was 15%, but in absolute terms smaller. In Figure 2B, the intersection between the pI of GOx (pI=4.2) and HFBI (pI = 5.7) is marked with a thick line. In the marked range, HFBI and GOx have opposite charges, whereas at more DOI: 10.1021/la101240e
8493
Article
Wang et al.
acidic conditions both will have positive charge, and at higher pH values both proteins are negatively charged. Adsorption of IgG, BSA, and Avidin to Surface Layers of HFBI and HGFI. Next we studied how IgG, BSA, and avidin bound to a preformed layer of both HFBI and HGFI. The data were collected as described above for GOx. Figure 3A shows how BSA binds to both HFBI and HGFI at different pH values. The binding of BSA was very similar to both hydrophobin layers and showed a very clear dependence on pH. In the plot of binding versus pH, there was a clear maximum peak of binding occurring around pH 4.5-6. The maximum binding of BSA is 300-350 ng/ cm2, which is in the range expected for a complete monolayer. Lines are drawn at the lower part of the graph to indicate the region of complementary charges according to pI values for BSA and hydrophobin (HFBI and HGFI). In Figure 3B, the corresponding binding curve of avidin to hydrophobin (HFBI and HGFI) is shown. This binding was clearly different from BSA, showing a much broader curve and with the binding clearly shifted toward higher pH values. At low pH there was almost no binding. The intersection of complementary charge according to pI are drawn at the lower part of the graph, showing again that maximum binding occurs in this region. In Figure 3C the binding of IgG on hydrophobin (HFBI and HGFI) is shown. The maximum binding occurred in the region around pH 7 with a clearly lower binding at high and low pH. The intersection showing complementary charge according to pI values are shown as thick lines at the lower part of the graph. Again, the highest binding was seen in this region. The dissipation values for avidin, IgG, and BSA binding to both hydrophobins were very low. The highest value was 1.1 10-6 for BSA on HFBI at pH 6.0. Otherwise, values were typically between 0.3 10-6 and 0.6 10-6, indicating very rigid layers. Adsorption of HFBI and HGFI on 1-Hexanethiol Surfaces at Different Ionic Strengths. The effect of ionic strength on the binding of both HFBI and HGFI to 1-hexanethiol was studied by increasing the molarity of the buffer solutions. Sodium acetate buffer pH 5.0 and sodium dihydrogen phosphate buffer at pH 7.0 were used at seven different concentrations, namely, 1, 50, 100, 200, 300, and 500 mM. The adsorbed amount of HFBI was not affected by increasing the ionic strength. The dissipation was very low, varying between 0.01 10-6 and 0.4 10-6. However, the ionic strength of the buffer affected HGFI to some extent. HGFI bound maximally (about 275 ng/cm2) at low buffer concentrations. When the ionic strength increased, the binding decreased almost linearly down to 170 ng/cm2 (data shown in Supporting Information Figure S1). For HGFI the dissipation varied between 0.5 10-6 and 1.8 10-6, indicating slightly less rigid films than measured for HFBI. The binding was very similar in both buffers tested. Adsorption of BSA, IgG, and Avidin on HFBI and HGFI at Different Ionic Strengths. To probe the nature of the binding between the anchoring hydrophobin layer and the second protein layer, we studied the effect of increasing ionic strength on binding. Experiments were performed with increasing concentration of sodium acetate pH 5.0 or sodium dihydrogen phosphate pH 7.0 as explained above for the free hydrophobins. In Figure 4 a representative example of the effect is shown for BSA binding to HFBI at pH 5.0. As can be seen, there is a clear decrease in binding as a function of the ionic strength of the buffer. The same general behavior was seen for the other proteins (IgG and avidin) at pH 7.0 and 5.0.
4. Discussion In this work we show that a surface bound layers of hydrophobin function as an adhesion layer onto which other proteins 8494 DOI: 10.1021/la101240e
Figure 4. Effect of ionic strength for the binding of BSA to hydrophobin surfaces. The x-axis shows the molarity of acetate buffer, pH 5.0. Higher ionic strength leads to a lower amount of bound BSA protein.
can bind. Two hydrophobins were studied: HFBI from T. reesei (belonging to class II) and HGFI from G. frondosa (class I). The proteins that were used for forming the second layer were BSA, monoclonal IgG, avidin, and GOx. They were chosen because they represent a range of different pI values, they are stable and soluble, and they are often used in surface binding studies because of their biotechnical applications. The surface onto which the hydrophobins were bound was a 1-hexanethiol layer on gold. This was chosen because hydrophobins readily bind to it, and it is easily prepared, uncharged, and nonpolar. Alternatively, silanized surfaces or spin-coated polymers could have been used. Hydrophobins first form a rigid monomolecular layer (Figure 1), and on top of this, a layer of other proteins can adsorb under suitable conditions. We suggest that this binding is mediated by ionexchange interactions. Our conclusion that hydrophobin forms a monomolecular layer derives from the calculation that the observed bound amount of 200 ng/cm2 corresponds to a mean area of 2.5 nm2 per molecule, which is in close agreement with the size of HFBI according to its X-ray structure.8 The calculation is not directly valid for HGFI since its exact structure is not known. However, since HFBI and HGFI are homologous, it is reasonable to assume that similar reasoning applies. Considering first GOx binding to HFBI, we note that the binding ability shows a clear maximum close to pH 5 (Figure 2B). Above pH 7 and below pH 3 there is practically no binding. We note that GOx has a pI of 4.219 and HFBI has a pI of 5.7.20 In Figure 2B, the pH range between these values is indicated by a thick line. In this range GOx and HFBI have opposite charges and show electrostatic attraction. Below and above, the molecules have the same charges and are expected to repel each other. The binding of BSA, avidin, and IgG follows the same general behavior (Figure 3). BSA has a pI of 4.5,21 avidin has a pI of 10.5,22 monoclonal IgG (IgG1-subclass) has a pI of 7-9.5,23 and HGFI has a pI of 3.3.16 HFBI and BSA are expected to have opposite (attractive) charges between pH 4.5 and 5.7. This is very close to the pH range at which adsorption was observed, with a (19) Pazur, J. H.; Kleppe, K. Biochemistry 1964, 3(4), 578–583. (20) Nakari-Set€al€a, T.; Aro, N.; Kalkkinen, N.; Alatalo, E.; Penttil€a, M. Eur. J. Biochem. 1996, 235(1-2), 248–255. (21) Hirayama, K.; Akashi, S.; Furuya, M.; Fukuhara, K. Biochem. Biophys. Res. Commun. 1990, 173(2), 639–646. (22) Marttila, A. T.; Airenne, K. J.; Laitinen, O. H.; Kulik, T.; Bayer, E. A.; Wilchek, M.; Kulomaa, M. S. FEBS Lett. 1998, 441(2), 313–317. (23) Prin, C.; Bene, M. C.; Gobert, B.; Montagne, P.; Faure, G. C. Biochim. Biophys. Acta 1995, 1243(2), 287–289.
Langmuir 2010, 26(11), 8491–8496
Wang et al.
Figure 5. Structure of HFBI showing the ionizable side chains of the protein. The protein binds to the solid surface through the hydrophobic patch (shown in green). In a dense surface packing it is likely that residues Asp30 and Lys32 are not solvent accessible and Asp40, Asp43, Arg 45, and Lys50 are exposed on the outer surface of the protein layer and are putatively responsible for the interactions described in this work.
slight shift toward higher pH values. For the HGFI surface the range of opposite charges would be between 3.3 and 4.5, and in this case, there is a clear shift in the binding toward higher pH. Possible explanations for this are discussed below. The binding of avidin to hydrophobin surfaces also follows the range of pH values between the pI values of the proteins where charges are complementary. The high pI of avidin suggests a maximum binding between pH 5.7 and 10.5 for the HFBI surface and for avidin of HGFI between 3.3 and 10.5. For IgG we see a maximum binding between the pI values for the hydrophobins and the IgG. We can conclude that all these cases where widely different proteins were used as probes follow closely the behavior expected from charge interactions. Another way of probing the nature of the interaction is to study the effect of increasing the ionic strength. In Figure 4, we show that binding decreases with increasing ionic strength as expected from charge interactions.24 Thus, four widely different proteins show a common binding behavior that is related to their pI values. The three-dimensional structure of HFBI (but not HGFI) is known.8 It contains three positively charged side chains (Lys32, Arg 45, Lys50) and three negatively charged side chains (Asp30, Asp40, Asp43). In addition, the amine N-terminus (Ser1) is positively charged and the carboxyl C-terminus (Ala75) is negatively charged. Interestingly, these charges are concentrated in the middle 20 residues of this 75 amino acid protein. The position of these residues in the three-dimensional structure is shown in Figure 5. For the function of HFBI, an important feature is its hydrophobic patch. This patch comprises about 19% of the surface of the protein and gives it an amphiphilic structure.8 In Figure 5 we show residues forming the hydrophobic patch in green. Previously, it has been shown that this patch is faced toward the solid surface when HFBI binds to a hydrophobic solid.11 In the figure, the protein is rotated so that all charged residues can be seen, but the view of the hydrophobic patch is not optimal. Four of the charged residues (Asp40, Asp43, Arg45, and Lys50) are located in the helix that is expected to be open toward the solution when HFBI is bound to a surface. We suggest that these residues are responsible for the ion-exchange behavior described in this work. Two of the charged residues (Asp30 and Lys32) are close to the hydrophobic patch and are probably not exposed to the solvent if HFBI is tightly packed on the surface. One possible function of these residues is that they may be involved in lateral (24) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1992; p 450.
Langmuir 2010, 26(11), 8491–8496
Article
protein-protein interaction. In the case of HFBI, the calculated pI allows a quite good explanation of its interactions with other proteins, as seen in Figures 2 and 3. However, the pI of HGFI does not explain its behavior equally well. The lower pI of HGFI suggests that its binding interactions would be shifted to lower pH values. However, because of effects of protein orientation resulting in the exposure of only some side chains, it is likely that the calculated pI in solution does not sufficiently describe the charge state of HGFI when bound on a surface. A proportionally larger fraction of the cationic side chains on critical solvent exposed parts of the protein would result in an apparently higher pI. This type of structural and conformation effect on ion-ion interactions is well-known from ion exchange chromatography.25,26 Structural effects may also affect the behavior of the second layer proteins, possibly explaining the slightly shifted binding of BSA to higher pH values. We also note that the mass of adsorbed HGFI is more variable than that of HFBI (Figure 1) which can affect the shape of the pH-dependent binding curves of the second protein. The binding of HGFI at different ionic strength is also more variable than the binding of HFBI (Figure S1). We can not with certainty explain the behavior of HGFI, but we find it very interesting that this class I hydrophobin behaves very similarly as the class II HFBI in binding a second layer, despite the abovementioned differences in adhesion. It is often experimentally observed that proteins have the lowest solubility around their pI value.27 This is generally seen as the proteins having a more hydrophobic character when they have the lowest number of charges. This aspect of solubility could be seen as an alternative explanation to the pH-dependent binding observed here. However, we note that all the proteins we used here are highly soluble and do not precipitate at the solution conditions and concentrations used here. The adhesion behavior was very reproducible and occurred for all proteins in a predictable way. The maximum binding did not occur at the pI of the proteins, but at pH values between the pI values of the hydrophobin and the adhering protein. Solubility should also decrease with increasing ionic strength, resulting in a higher binding at high salt, but the opposite was seen here (Figure 4). Precipitated protein should also result in high dissipation values, but overall only low dissipation values were observed. We therefore conclude that it is very unlikely that our results could be explained by reduced solubility of the adhering protein. We studied the functionality of the surface bound IgG by injecting its antigen (C-reactive protein) over it and concluded that it had retained its binding capacity very well (data not shown). Other reports also confirm that protein adhesion to a hydrophobin layer results in a well retained functionality.12,14,28-31 However, as shown in the present work, the adhesiveness is based on ion-exchange interactions, and therefore it will always be unpredictable in which orientation the second layer of protein is bound. Nonetheless, it seems that even if orientation has not been controlled in these experiments, the binding orientation has been (25) Malmquist, G.; Nilsson, U. H.; Norrman, M.; Skarp, U.; Str€omgren, M.; Carredano, E. J. Chromatogr. A 2006, 1115(1-2), 164–186. (26) Yamamoto, S.; Fujii, S.; Yoshimoto, N.; Akbarzadehlaleh, P. J. Biotechnol. 2007, 132(2), 196–201. (27) Arakawa, T.; Timasheff, S. N. Theory of protein solubility. In Methods in Enzymology; Academic Press: New York, 1985; Vol. 114, pp 49-77. (28) Corvis, Y.; Brezesinski, G.; Rink, R.; Walcarius, A.; Van der Heyden, A.; Mutelet, F.; Rogalska, E. Anal. Chem. 2006, 78(14), 4850–4864. (29) Corvis, Y.; Trzcinska, K.; Rink, R.; Bilkova, P.; Gorecka, E.; Bilewicz, R.; Rogalska, E. J. Phys. Chem. C 2007, 111(3), 1176–1179. (30) Corvis, Y.; Walcarius, A.; Rink, R.; Mrabet, N. T.; Rogalska, E. Anal. Chem. 2005, 77(6), 1622–1630. (31) Zhao, Z. X.; Qiao, M. Q.; Yin, F.; Shao, B.; Wu, B. Y.; Wang, Y. Y.; Wang, X. S.; Qin, X.; Li, S.; Yu, L.; Chen, Q. Biosens. Bioelectron. 2007, 22(12), 3021– 3027.
DOI: 10.1021/la101240e
8495
Article
favorable. We can expect that the underlying hydrophobin layer does not easily cause denaturation of the bound second layer. Denaturation can often be seen when proteins bind to surfaces such as polystyrene, but we can expect that a protein surface is more compatible and would not lead to denaturation. This has been observed when developing immunoassays using multilayer techniques.32,33 In conclusion, we here present a mechanism why and how hydrophobins function as a “primer” layer for protein adhesion. We suggest that the interactions between the hydrophobin and the second layer are due to electrostatic interactions. The results show that we can predict the adsorption behavior of different proteins on surface-bound hydrophobins if the pI of the adsorbing protein is known. The similarity in function of the two different hydrophobins is striking and presents an interesting problem that (32) Davies, J.; Roberts, C. J.; Dawkes, A. C.; Sefton, J.; Edwards, J. C.; Glasbey, T. O.; Haymes, A. G.; Davies, M. C.; Jackson, D. E. Langmuir 1994, 10 (8), 2654–2661. (33) Suter, M.; Butler, J. E.; Peterman, J. H. Mol. Immunol. 1989, 26(3), 221– 230.
8496 DOI: 10.1021/la101240e
Wang et al.
can teach us more about the biological roles of hydrophobins. Overall, this work will be important for understanding more details of the molecular mechanisms of the biological function of hydrophobins and may also lead to improved biotechnical applications. Acknowledgment. Tuja Teerinen is thanked for help with the CRP detection, and Riitta Suihkonen is thanked for technical assistance. Dr. Vesa Hytonen is thanked for the donation of avidin. M.B.L., M.L., and Z.W. acknowledge the support of the Academy of Finland (Grants #118519 and #131055). Z.W. and M.Q. acknowledges the program for new century excellent talents in university (Grant #NCET-06-0212), and the Sino-Finnish scientific and technological cooperation project from the Ministry of Science and Technology of China (Grant #2006DFA32360). Supporting Information Available: Figure S1. This material is available free of charge via the Internet at http://pubs. acs.org.
Langmuir 2010, 26(11), 8491–8496
