Self-Assembled Films of Hydrophobin Proteins HFBI and HFBII

Dec 18, 2008 - Xiaoli L. Zhang , Jeffrey Penfold , Robert K. Thomas , Ian M. Tucker , Jordan T. Petkov , Julian Bent , Andrew Cox , and Richard A. Cam...
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Langmuir 2009, 25, 1612-1619

Self-Assembled Films of Hydrophobin Proteins HFBI and HFBII Studied in Situ at the Air/Water Interface Kaisa Kisko,*,† Ge´za R. Szilvay,‡,§ Elina Vuorimaa,¶ Helge Lemmetyinen,¶ Markus B. Linder,‡ Mika Torkkeli,† and Ritva Serimaa† DiVision of Materials Physics, Department of Physics, UniVersity of Helsinki, POB 64, FI-00014, Finland, VTT Biotechnology, POB 1500, FI-02044 VTT, Finland, and Department of Chemistry and Bioengineering, Tampere UniVersity of Technology, POB 541, FI-33101 Tampere, Finland ReceiVed October 3, 2008. ReVised Manuscript ReceiVed NoVember 18, 2008 Hydrophobins are a group of surface-active fungal proteins known to adsorb to the air/water interface and selfassemble into highly crystalline films. We characterized the self-assembled protein films of two hydrophobins, HFBI and HFBII from Trichoderma reesei, directly at the air/water interface using Brewster angle microscopy, grazingincidence X-ray diffraction, and reflectivity. Already in zero surface pressure, HFBI and HFBII self-assembled into micrometer-sized rafts containing hexagonally ordered two-dimensional crystallites with lattice constants of 55 Å and 56 Å, respectively. Increasing the pressure did not change the ordering of the proteins in the crystallites. According to the reflectivity measurements, the thicknesses of the hydrophobin films were 28 Å (HFBI) and 24 Å (HFBII) at 20 mN/m. The stable films could also be transferred to a silicon substrate. Modeling of the diffraction data indicated that both hydrophobin films contained six molecules in the unit cell, but the ordering of the molecules was somewhat different for HFBI and HFBII, suggesting specific protein-protein interactions.

1. Introduction Proteins contain typically both hydrophobic and hydrophilic amino acids, which enables their adsorption to hydrophobic/ hydrophilic interfaces, such as the air/water interface.1,2 The adsorption depends on the extent of exposure of the hydrophobic side chains and therefore tends to lead to some degree of conformational change in the proteins. For rigid globular proteins, large conformational changes may be needed,3,4 which makes the adsorption irreversible. More flexible proteins, such as the naturally disordered β-casein, can adopt various conformations more easily, and therefore, the adsorption is more reversible.3,5 Hydrophobins are a group of very surface-active proteins, which readily adsorb to various surfaces and interfaces.6,7 According to protein crystallography results, there is a hydrophobic patch consisting of alipathic side chains on the surface of hydrophobin proteins HFBI8 and HFBII9 from Trichoderma reesei. Therefore, these proteins can adsorb to the air/water interface without changing the secondary structure.10 At the * Corresponding author. E-mail: [email protected]. † University of Helsinki. ‡ VTT Biotechnology. § Current address: Columbia University, 500 W 120th Street, NY 10027, USA. ¶ Tampere University of Technology.

(1) Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110–115. (2) Murray, B. S. Curr. Opin. Colloid Interface Sci. 2007, 12, 232–241. (3) Freer, E. M.; Yim, K. S.; Fuller, G. G.; Radke, C. J. Langmuir 2004, 20, 10159–10167. (4) Lad, M. D.; Birembaut, F.; Matthew, J. M.; Frazier, R. A.; Green, R. J. Phys. Chem. Chem. Phys. 2006, 8, 2179–2186. (5) Martin, A. H.; Meinders, M. B. J.; Bos, M. A.; Cohen Stuart, M. A.; van Vliet, T. Langmuir 2003, 19, 2922–2928. (6) Wo¨sten, H. A. B.; Schuren, F. H. J.; Wessels, J. G. H. EMBO J. 1997, 38, 363–374. (7) Linder, M. B.; Szilvay, G. R.; Nakari-Seta¨la¨, T.; Penttila¨, M. E. FEMS Microbiol. ReV. 2005, 29, 877–896. (8) Hakanpa¨a¨, J.; Szilvay, G. R.; Kaljunen, H.; Maksimainen, M.; Linder, M.; Rouvinen, J. Protein Sci. 2006, 15, 2129–2140. (9) Hakanpa¨a¨, J.; Paananen, A.; Askolin, S.; Nakari-Seta¨la¨, T.; Parkkinen, T.; Penttila¨, M.; Linder, M. B.; Rouvinen, J. J. Biol. Chem. 2004, 279, 534–539. (10) Askolin, S.; Linder, M.; Scholtmeijer, K.; Tenkanen, M.; Penttila¨, M.; de Vocht, M.; Wo¨sten, H. A. B. Biomacromolecules 2006, 7, 1295–1301.

interface, hydrophobins self-assemble into highly ordered films and lower the surface tension of water; for example, HFBII lowers it from 72 mJ/m2 to 28 mJ/m2.10 The self-assembled hydrophobin films at the air/water interface have a very high surface shear elasticity, and they stabilize air bubbles and foams.11 In addition, hydrophobins self-assemble at interfaces between oil and water6,12 and a hydrophobic solid and water.6 Hydrophobins are small, ca. 7-10 kDa proteins originating from filamentous fungi.7 The primary structure is characterized by a conserved pattern of eight cysteine residues, which make four intramolecular disulfide bridges.7 Hydrophobins have multiple roles in fungal development, most of which utilize the self-assembled films. The hydrophobin films at the air/water interface lower the surface tension of water, thereby enabling the growth of aerial structures.13 Hydrophobin films also coat spores14 and aerial hyphae,15 and mediate the attachment of the fungus to solid surfaces.6 The amphiphilic structure of the hydrophobin monomers, responsible for their interfacial behavior, also leads to association of hydrophobins in aqueous solution. At low concentrations, the proteins can exist as monomers, but the hydrophobic effect drives hydrophobins to form assemblies at higher concentrations.16,17 For example, for the T. reesei hydrophobins HFBI and HFBII tetramers are the dominant assemblies in solution at mg/mL concentrations.17-19 The exact relationship between the solution (11) Cox, A. R.; Cagnol, F.; Russell, A. B.; Izzard, M. J. Langmuir 2007, 23, 7995–8002. (12) Wang, X.; Shi, F.; Wo¨sten, H. A. B.; Hektor, H.; Poolman, B.; Robillard, G. T. Biophys. J. 2005, 88, 3434–3443. (13) Wo¨sten, H. A. B.; van Wetter, M.-A.; Lugones, L. G.; van der Mei, H. C.; Busscher, H. J.; Wessels, J. G. H. Microbiology 2000, 146, 767–773. (14) Stringer, M. A.; Dean, R. A.; Sewall, T. C.; Timberlake, W. E. Genes DeV. 1991, 5, 1161–1171. (15) Wo¨sten, H. A. B.; de Vries, O. M. H.; Wessels, J. G. H. Plant Cell 1993, 5, 1567–1574. (16) Wang, X.; Graveland-Bikker, J. F.; de Kruif, C. G.; Robillard, G. T. Protein Sci. 2004, 88, 810–821. (17) Szilvay, G. R.; Nakari-Seta¨la¨, T.; Linder, M. B. Biochemistry 2006, 45, 8590–8598. (18) Torkkeli, M.; Serimaa, R.; Ikkala, O.; Linder, M. Biophys. J. 2002, 83, 2240–2247.

10.1021/la803252g CCC: $40.75  2009 American Chemical Society Published on Web 12/18/2008

Self-Assembled Films HFBI and HFBII

assemblies and self-assembled films has remained elusive, partially due to lack of molecular-level information on the selfassembled films. In a recent study,20 the interfacial behavior of native HFBI was compared to an engineered HFBI variant that in solution formed native-like tetramers, but unlike native HFBI, did not dissociate even below µg/mL. Both proteins adsorbed to the air/water interface and lowered the surface tension of water in a similar way, indicating that the dissociation into monomers is not necessary for adsorption. There is very little in situ information on the self-assembled hydrophobin films at the air/water interface. Studies using infrared21 and circular dichroism10 spectroscopies have probed the secondary structures of the proteins at the air/water interface, but unfortunately they do not give information on the arrangement of the proteins in the films. In another approach, the self-assembled films at the air/water interface have been transferred to solid substrates and studied using electron21 or atomic force microscopy (AFM).22,23 In particular, in a recent study23 a self-assembled film of hydrophobin HFBI was transferred to a graphite substrate using the Langmuir-Schaefer (LS) technique, and thereafter kept and imaged in water. The film was highly crystalline and had a thickness of 28 Å. In dried Langmuir-Blodgett (LB) films, a thickness of only 13 Å was observed,22,23 highlighting the importance of in situ studies. Here, we have studied the self-assembled hydrophobin protein films directly at the air/water interface using Brewster angle microscopy (BAM), grazing-incidence X-ray diffraction (GID), and reflectivity. BAM is sensitive to local changes in the optical properties of the interface and can probe micrometer-sized areas. It is particularly useful for studying monolayer phase transitions, since one can discern among changes in packing density, longrange ordering, or film thickness between coexisting phases. X-ray reflectivity and GID, on the other hand, probe the nanometer length scales in the protein films. Reflectivity gives information on the eletron density profile of the film, while GID is used to probe the ordering of the hydrophobin molecules. GID is frequently used to study Langmuir films of long-chain fatty acids,24 where it can provide near-atomic resolution structural information.25 The technique has also been applied to a few protein films at the air/water interface,26-31 but the resolution has been limited by the difficulty of obtaining perfect enough two-dimensional protein crystals.29,31 The proteins studied at the air/water interface also include the hydrophobin HFBIII from T. reesei, which was shown to form two-dimensional, hexagonally (19) Kisko, K.; Szilvay, G. R.; Vainio, U.; Linder, M. B.; Serimaa, R. Biophys. J. 2008, 94, 198–206. (20) Szilvay, G. R.; Kisko, K.; Serimaa, R.; Linder, M. B. FEBS Lett. 2007, 581, 2721–2726. (21) de Vocht, M. L.; Reviakine, I.; Ulrich, W.-P.; Bergsma-Schutter, W.; Wo¨sten, H. A. B.; Vogel, H.; Brisson, A.; Wessels, J. G. H.; Robillard, G. T. Protein Sci. 2002, 11, 1199–1205. (22) Paananen, A.; Vuorimaa, E.; Torkkeli, M.; Penttila¨, M.; Kauranen, M.; Lemmetyinen, H.; Serimaa, R.; Linder, M. B. Biochemistry 2003, 42, 5253–5258. (23) Szilvay, G. R.; Paananen, A.; Laurikainen, K.; Vuorimaa, E.; Lemmetyinen, H.; Peltonen, J.; Linder, M. B. Biochemistry 2007, 46, 2345–2354. (24) Als-Nielsen, J.; Jacquemain, D.; Kjaer, K.; Leveiller, F.; Lahav, M.; Leiserowitz, L. Phys. Rep. 1994, 246, 251–313. (25) Pignat, J.; Daillant, J.; Leiserowitz, L.; Perrot, F. J. Phys. Chem. B 2006, 110, 22178–22184. (26) Haas, H.; Brezesinski, G.; Mo¨hwald, H. Biophys. J. 1995, 68, 312–314. (27) Verclas, S. A.; Howes, P. B.; Kjaer, K.; Wurlitzer, A.; Weygand, M.; Bu¨ldt, G.; Dencher, N. A.; Lo¨sche, M. J. Mol. Biol. 1999, 287, 837–843. (28) Weygand, M.; Wetzer, B.; Pum, D.; Sleytr, U. B.; Cuvillier, N.; Kjaer, K.; Howes, P. B.; Lo¨sche, M. Biophys. J. 1999, 76, 458–468. (29) Lenne, P.-F.; Berge, B.; Renault, A.; Zakri, C.; Ve´nien-Bryan, C.; Courty, S.; Balavoine, F.; Bergsma-Schutter, W.; Brisson, A.; Gru¨bel, G.; Boudet, N.; Konovalov, O.; Legrand, J. F. Biophys. J. 2000, 79, 496–500. (30) Kisko, K.; Szilvay, G. R.; Vuorimaa, E.; Lemmetyinen, H.; Linder, M. B.; Torkkeli, M.; Serimaa, R. J. Appl. Crystallogr. 2007, 40, s355–s360. (31) Miller, C. E.; Majewski, J.; Watkins, E. B.; Weygand, M.; Kuhl, T. L. Biophys. J. 2008, 95, 641–647.

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ordered crystallites with lattice constant a ) 56 Å. The interpretation of the results was hampered by the lack of information on the folded structure of the hydrophobin, but a model where the proteins formed helical rings consisting of six protein molecules was suggested.30 We concentrate on the two more thoroughly studied T. reesei hydrophobins, HFBI and HFBII. They have slightly different roles in the fungus,32 as HFBI is found from the cell walls33 and HFBII on the spores.34 However, the folded structures of HFBI and HFBII are very similar,8,9 as is their behavior both in solution17-20 and on solid substrates.18,22,23,35,36 Here, we characterize the self-assembled films of the proteins in situ at the air/water interface.

2. Materials and Methods 2.1. Protein Production and Purification. HFBI and HFBII were expressed and purified as described previously.22 2.2. Surface Pressure-Area Isotherms and Brewster Angle Microscopy. The full isotherms and Brewster angle microscope images were measured simultaneously at the Department of Chemistry and Bioengineering, Tampere University of Technology, with a KSV OPTREL BAM300 mounted on a KSV minitrough Langmuir film balance (KSV Instruments) equipped with a double barrier system. A 1 mM acetate buffer (pH ) 5.0) in water purified by a Milli-Q system (Millipore) with resistivity greater than 18.0 MΩ cm was used as a subphase. The temperature of the subphase was 18.0 ( 0.5 °C, and the compression speed was 4.5 cm2/min. 200 µL of the 30 µM hydrophobin solution was spread at the air/water interface and allowed to stabilize for 20 min before compression. The instrument is equipped with a 10 mW HeNe laser (633 nm) linearly polarized in the plane of incidence by a Glann-Thomson polarizer. The reflection from the interface passes through a second Glann-Thomson polarizer and is picked by a CCD camera. The spatial resolution of the system is approximately 2 µm. The image processing procedure included a geometrical correction of the image as well as a filtering operation to reduce noise. Furthermore, the brightness of each image was scaled to improve contrast. 2.3. Grazing-Incidence X-ray Diffraction and Reflectivity. The grazing-incidence X-ray diffraction and reflectivity measurements were conducted at the beamline ID10B at the European Synchrotron Radiation Facility. A Ge(111) deflector crystal was used to deflect the incoming beam down from the horizontal direction. The beam was monochromatized using a diamond (111) double crystal monochromator to the wavelength λ ) 1.55 Å. The beam size was reduced by slits to be 0.1 mm vertically and 0.5 mm (HFBII in 1 mM acetate subphase) or 1 mm horizontally. The measurements at the air/water interface were conducted in a 170 × 438 mm Langmuir trough with a single moving barrier. The surface pressure was measured using a Wilhelmy balance. The trough was mounted on active vibration isolation system (MOD-2 S, Halcyonics). A 1 mM or 50 mM acetate buffer (pH ) 5.0) in water purified by a Milli-Q system (Millipore) with resistivity greater than 18.2 MΩ cm was used as a subphase. (32) Askolin, S.; Penttila¨, M.; Wo¨sten, H. A. B.; Nakari-Seta¨la¨, T. FEMS Microbiol. Lett. 2005, 253, 281–288. (33) Nakari-Seta¨la¨, T.; Aro, N.; Kalkkinen, N.; Alatalo, E.; Penttila¨, M. Eur. J. Biochem. 1996, 235, 248–255. (34) Nakari-Seta¨la¨, T.; Aro, N.; Ilme´n, M.; Mun˜oz, G.; Kalkkinen, N.; Penttila¨, M. Eur. J. Biochem. 1997, 248, 415–423. (35) Ritva, S.; Torkkeli, M.; Paananen, A.; Linder, M.; Kisko, K.; Knaapila, M.; Ikkala, O.; Vuorimaa, E.; Lemmetyinen, H.; Seeck, O. J. Appl. Crystallogr. 2003, 36, 499–502. (36) Kisko, K.; Torkkeli, M.; Vuorimaa, E.; Lemmetyinen, H.; Seeck, O. H.; Linder, M.; Serimaa, R. Surf. Sci. 2005, 584, 35–40.

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The temperature of the subphase was 21.5 ( 0.5 °C or 20.2 ( 0.3 °C (HFBII in 1 mM subphase). The 40 µM (HFBI) or 30 µM (HFBII) hydrophobin solutions were allowed to incubate for at least 24 h before spreading on the subphase. 500 µL (HFBI in 1 mM subphase), 570 µL (HFBI in 50 mM subphase), or 560 µL (HFBII) of the protein solution was spread on the subphase. The trough was covered, and helium flow into the trough was connected. The compression was started once the oxygen content of air fell below 0.5%, typically 20 min after the injection. The equilibrium pressures at the start of the measurements were 0 mN/m (HFBI) and 0 mN/m (HFBII) at the mean molecular areas of 514 Å2 (HFBI) and 699 Å2 (HFBII) in 1 mM subphase. For grazing-incidence measurements, the angle of incidence Ri was chosen to be 0.11° or 0.12° (HFBII in 1 mM subphase). The intensities were recorded using a Soller collimated 50 mm MBraun linear position-sensitive detector. The measurements were made in scattering angle range 2θ ) 1.5-10° with a step of 0.025°. The range of the vertical exit angle Rf was Rf ) 0-12°. Measurement times were typically 7 s/point, and after each movement of the detector, the system was allowed to stabilize for 3 s. The reflectivity measurements were conducted at an angular range of 0 eRi ) Rf e 5°. All the results are presented in terms of the scattering vector q, where |q| ) 4π sin θ/λ. After the GID measurement at the lowest pressure, the same HFBII sample was compressed to pressures 1, 5, 10, 15, 20, and 30 mN/m and GID was measured at each pressure. The compression speed was 17 cm2/min. Before starting the measurements at each pressure, the system was allowed to stabilize for 5 min. The reflectivity scans were taken separately, from a different sample at pressures 10 and 20 mN/m. For HFBI, the GID and reflectivity were measured from the same sample in the following sequence: Π ) 0 mN/m, GID and reflectivity; Π ) 13 mN/m, reflectivity; Π ) 20 mN/m, reflectivity and GID; Π ) 30 mN/m, GID and reflectivity. In a repeat measurement from a different purification set, GID was measured at pressures 2.3, 5, 10, and 13 mN/m, and the results were the same. Both proteins were also studied in 50 mM subphase, where they were directly compressed to 20 mN/m and both reflectivity and GID were measured. The mean molecular areas at the start of these measurements were 327 Å2 (HFBI) and 373 Å2 (HFBII). After measurements at 13 mN/m for HFBI and 30 mN/m for HFBII, the protein films were lifted to a silicon substrate using the LS technique, i.e., by bringing the substrate horizontally into contact with the protein layer at the air/water interface. The LS films were dried in vacuum.

3. Results 3.1. Air/Water Interface: Brewster Angle Microscopy. Both of the studied T. reesei proteins, HFBI and HFBII, adsorb to the air/water interface and self-assemble into films. The formation of the films was followed using BAM at micrometer-length scales. After spreading, the film was allowed to stabilize for 20 min before compression was started. During this time, for HFBI mainly pure subphase surface was observed with thin rafts (Figure 1A) floating in and out of the view of the camera. The size of these rafts was larger than the view of the camera (400 × 600 µm2), and they consisted of domains smaller than the resolution (2 µm) of the camera. For HFBII, on the other hand, the surface was covered with small domains (Figure 1B) already directly after spreading. The structures seem less ordered and their borders less defined than for HFBI. This is probably due to constant reorganization taking place in the film. Upon compression (Supporting Information Figure S1A-N) from 1 mN/m onward, the surfaces were fully covered with the

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Figure 1. Brewster angle microscopy images of (A) HFBI and (B) HFBII rafts at the air/water interface taken 6 and 5 min after injection. The surface pressures are Π ) 0.03 mN/m and Π ) 0.16 mN/m with corresponding mean molecular areas of 1001 Å2 and 901 Å2, respectively. The sizes of the images are 170 × 108 µm2.

film for both hydrophobins. Increasing the pressure led to an increase in the brightness of the figures, indicating increase in the thickness of the film. For HFBI, the fine structure observed at lower pressures has almost disappeared at 16 mN/m (Figure S1I) indicating that there is very little variation in the film thickness. The smoothest film is observed at 22 mN/m (Figure S1K). At 30 mN/m (Figure S1M), circles with ripples appear, indicating the beginning of collapse. For HFBII, the film is smoothest at 30 mN/m (Figure S1N), and the indications of collapse do not start before 40 mN/m. For HFBI, the brightness increases clearly from 0 to 16 mN/ m, and only small changes are observed at higher pressures. For HFBII, the brightness changes are smaller and continue until 30 mN/m. This indicates that the overall film thickness changes slightly but continuously during compression until 30 mN/m is reached. It seems that for HFBII the small domains persist to higher surface pressures before finally merging to a uniform layer. 3.2. Air/Water Interface: Grazing-Incidence Diffraction. The self-assembled HFBI and HFBII rafts at the air/water interface were probed using GID to obtain information on the ordering of the protein molecules in the crystalline parts of the films. The first measurements at zero surface pressure (maximum area of the trough) were started once the oxygen level had decreased enough, typically 20 min after the injection. According to repeated GID measurements, there were no major changes in the crystal structure of the films during the 1 h measurements. The changes in the mean molecular areas were less than 10 Å2. The mean molecular areas at each pressure are shown in Figure 2 with the full isotherms of HFBI and HFBII.

Self-Assembled Films HFBI and HFBII

Figure 2. Full isotherms of (solid line) HFBI and (dashed line) HFBII obtained during the BAM measurements. The mean molecular areas at each pressure before, between, and after the GID and reflectivity measurements for (squares) HFBI and (diamonds) HFBII. The average changes in the mean molecular areas during the different measurements were 5 Å2 and 6.5 Å2 for HFBI and HFBII, respectively. The subphase was 1 mM acetate buffer in all measurements.

Figure 3. The two-dimensional diffraction patterns of (A) HFBI and (B) HFBII at Π ) 20 mN/m in 50 mM subphase. The background has not been subtracted, and the maxima at the smallest q-values close to the primary beam arise from the setup.

The grazing-incidence diffraction results from the selfassembled HFBI and HFBII films are presented in Figures 3, 4, and 5. Figure 3 shows the two-dimensional diffraction patterns of (A) HFBI and (B) HFBII at surface pressure 20 mN/m in 50 mM subphase. The two-dimensional data clearly show diffraction maxima arising from the crystalline parts of the films at the air/water interface. These crystalline parts might correspond to the domains seen in the BAM images, but there is no clear

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Figure 4. The integrated intensities of HFBI and HFBII as a function of pressure in 1 mM subphase. The integration is over the whole vertical q-range (as seen in Figure 3). The solid curves are measured in 1 mM subphase and the dotted in 50 mM subphase. The HFBI LS film was prepared separately, while the LS film of HFBII was lifted after the measurement at 30 mN/m. For the LS films, the integration is over the vertical q-range 0-0.1 1/Å. The curves have been vertically shifted for clarity, and the background has not been subtracted.

evidence for that. On the basis of the BAM results, the sizes of the crystalline areas are much smaller than the footprint of the beam on the sample, so that the crystallites form a two-dimensional powder. The positions of the intensity maxima in the horizontal direction indicate a hexagonal ordering in the lateral direction. The intensity distributions of the maxima in the vertical direction, along the Bragg rods, give structural information on the direction perpendicular to the film surface. Figure 4 shows the integrated intensities of HFBI and HFBII as a function of pressure. The integration is perfomed over the whole vertical q-range in Figure 3. Already, the first GID measurements at zero surface pressure contain the intensity maxima arising from a hexagonal lattice with unit cell parameters of a ) b ) 55 Å, γ ) 120° (HFBI) and a ) b ) 56 Å, γ ) 120° (HFBII). As the pressure is increased, the unit cell shrinks slightly, to a ) 54 Å (HFBI) and a ) 55 Å (HFBII). The integrated intensities vary a little. For HFBI, the integrated intensity increases from 0 to 20 mN/m, but decreases from 20 to 30 mN/m. For HFBII, the integrated intensities do not behave in a systematic manner, but the highest intensity is obtained at the highest measurement pressure, 30 mN/m. The increase in intensity with surface pressure can be explained by assuming that the individual crystallites are forced closer together as the available area on the Langmuir trough decreases and therefore cover the surface more uniformly. Unfortunately, the widths of the peaks are too close to the instrumental resolution to allow a reliable determination of the sizes of the crystalline domains. The intensity variations along the first four Bragg rods, indexed as 10, 11, 20, and 21, are shown in Figure 5A for HFBI and Figure 5B for HFBII. The profiles of the 10 peak of the two proteins differ markedly, while the other profiles have identical shapes. This indicates that the packing of the molecules in the unit cells is slightly different for HFBI and HFBII. The intensity distributions along the peaks do not change as a function of pressure, further proving that the arrangement of the proteins within the unit cell does not change as the pressure is increased. 3.3. Air/Water Interface: Reflectivity. The reflectivity measurements were used to obtain information on the thickness of the protein film, on its electron density profile, and on the roughnesses of the interfaces between air and film, and film and water. Figure 6 shows the reflectivity curves of HFBI and HFBII

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Figure 6. Reflectivity curves of HFBI and HFBII as a function of pressure. The solid curves are measured in 1 mM subphase and the dotted in 50 mM subphase. The reflectivity curve of the pure 1 mM subphase is also shown.

Figure 7. The reflectivity curves of HFBI and HFBII at Π ) 20 mN/m in 50 mM subphase divided by the reflectivity curve of 1 mM subphase. The solid (dashed) lines are the two-box (one-box) models fitted to the experimental curves. The electron density distributions of the models are shown in the inset. Figure 5. The integrated intensities along the Bragg rods at Π ) 20 mN/m in 50 mM subphase for (A) HFBI and (B) HFBII with models below. The experimental data are shown as dots, and the calculated intensities of the models as solid lines. The background, taken from the same data around each rod, has been subtracted. The maximum intensity of each rod has been scaled to unity, and the curves have been shifted for clarity. The corresponding sphere models are represented below for HFBI (left column) and HFBII (right column). Each sphere represents the position of a hydrophobin molecule. The dashed lines mark the unit cell. For HFBI, some of the dimers are connected for clarity. The lighter spheres are 20 Å (HFBI) or 21 Å (HFBII) below their darker dimer pairs. See the text for more details.

as a function of pressure. The reflectivity of HFBI at 0 mN/m is featureless, but in all of the other curves, oscillations arising from the protein film can be distinguished. Increasing pressure affects the HFBI films only slightly, while for HFBII, a more significant effect is seen. To highlight the weak oscillations in the reflectivity curves, Figure 7 shows the reflectivity curves of HFBI and HFBII at Π ) 20 mN/m divided by the reflectivity of the subphase. The data treated this way were used in the modeling of the reflectivity. All the data were rather smooth and contained only a few almost evenly spaced maxima. Therefore, they were described using the box-model, where one to three boxes of constant electron density were used to describe the protein film. Here, it should

be noted that using, for example, two boxes to describe the film does not indicate that there is a double layer of proteins, but only that the electron density within the film is not constant. The box edges were smoothed by Gaussian functions, and the resulting reflectivity curves were calculated in the Born approximation.24 In Figure 7, the experimental reflectivity curves of HFBI and HFBII are shown together with fitting results from one- and two-box models. The one-box model describes the HFBI film adequately, and the improvement gained by adding a second box is small. In both one- and two-box models, the total thickness of the film is 28 Å. The surface pressure has only a small effect on the reflectivity pattern of the HFBI film. In the pressure series, measured in the 1 mM buffer, the position of the second minimum shifts to slightly lower q-values as the pressure increases. Therefore, these data are better fitted with a two-box model (data not shown). The position of the first minimum, however, does not change, and the total thickness of the film stays at 28 Å. For HFBII, the change of the film thickness with pressure is more pronounced, and can be clearly seen also in the initial reflectivity curve in Figure 6. At 10 mN/m, the thickness of the film is about 28 Å. However, at 20 mN/m the thickness has decreased to 24 Å. The GID patterns do not indicate a change in the crystalline ordering of the film from 10 to 20 mN/m.

Self-Assembled Films HFBI and HFBII

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Therefore, the change in the thickness might be due to the noncrystalline material at the air/water interface. 3.4. Modeling of the GID Results at the Air/Water Interface. The intensity distributions of the Bragg rods (Figure 5A,B) are very clear, and therefore allow an attempt to elucidate the positions of the individual hydrophobin molecules inside the unit cell. We follow the strategy commonly used to model Langmuir films of long-chain fatty acids and model the protein film ignoring the water, as if it were in vacuum. The intensity profiles of the Bragg rods hk are calculated using the structure factor25,37

Fhk(qz) )

∑ fj exp[2πi(hxj + kyj) + iqzzr,j]

(1)

j

where fj is the atomic form factor of atom j situated at (xj, yj, zr, j/c) within the unit cell coordinates. The atomic structures of the folded monomers of HFBI8 and HFBII9 in the single crystals were taken from the protein crystallography data. The monomers were rotated to have their hydrophobic patches on top. Model systems were generated in hexagonal space groups using both 3 and 6 monomers per unit cell. The films were one unit cell thick. Unfortunately, these models did not describe the data adequately. For comparison, the monomers of HFBI and HFBII were also represented by spheres. In these models, the oscillations along the Bragg rods arise from the (relative) heights of the spheres and are modified by the form factor of the spheres. The fitted radii of the spheres were 8 Å for both proteins, which is somewhat smaller than expected for the rather spherical molecules with radii of gyrations between 11 and 14 Å.8,9 For both proteins, six spheres per unit cell was found to be the best arrangement. In many of the models, two spheres were close to each other, so as to create dimers. Amphiphilic dimers have been observed also in one crystal structure of HFBII.38 Therefore, more dimeric models were tested. Figure 5 shows, for example, such dimeric models for HFBI and HFBII in space group 144 (P31). For HFBI, the first sphere of the dimer is located at (x, y) ) (0.45, 0.25), and the second 21 Å above the first at (x - 0.2, y + 0.25). The dimers are quite loose, as the distance between the centers of the spheres in the dimers is 29 Å. However, this is of the same order of magnitude as the longest distances within the actual molecules. The helical rise perpendicular to the film surface is 4 Å, and the other two dimers are created by the symmetry operations of the space group, so that the second dimer is 4 Å and the third 8 Å above the first one. The arrangement leads to formation of two trimers, a tightly packed trimer on the lower and a more loosely packed trimer on the upper level. The model describes the data well at the lowest q-values and, for example, reproduces the minimum to the 10 peak at q ) 0 1/Å. However, the form factor of the spheres effectively dampens the oscillations at the higher q-values, and therefore, the current model cannot fit, for example, the second maximum of the 10 peak. For HFBII, the correspondence between the data and the model is somewhat better. In the HFBII model, the first sphere of the dimer is located at (x, y) ) (0.15, 0.35) and the second 20 Å above the first at (x + 0.05, y + 0.1). The helical rise perpendicular to the film surface is 3 Å, so the total film thickness from the center of the lowest sphere to the center of the highest sphere is 26 Å. The dimers are slightly tilted in such a way that, when looking from below, the lower spheres of the dimers seem to (37) Kmetko, J.; Datta, A.; Evmenenko, G.; Dutta, P. J. Phys. Chem. B 2001, 105, 10818–10825. (38) Kallio, J. M.; Linder, M. B.; Rouvinen, J. J. Biol. Chem. 2007, 282, 28733–28739.

form a tight trimer. Looking from above, the upper spheres are further away from each other and create six sphere rings. This kind of difference between upper and lower surfaces of hydrophobin films was found in the AFM experiments of HFBI LS and LB films.23 When comparing the HFBI and HFBII model films, it should be noted that they have almost the same density. Both contain six molecules per unit cell, but the packing is different. 3.5. Dried LS Films: Ordering. After the measurement at the air/water interface at the final pressure (13 mN/m for HFBI and 30 mN/m for HFBII), the hydrophobin films were manually transferred to silicon substrate using the LS technique. Figure 4 shows the integrated intensity of the HFBII sample (HFBI not shown) and an LS film of HFBI prefabricated at Tampere University of Technology (see section 2.2). The GID patterns confirm the success of the transfers: The positions of the intensity maxima are the same at the air/water interface and on the solid substrate, meaning that the hexagonal ordering and the sizes of the unit cells remain the same. However, the peaks in the transferred films are rather faint and the intensity distributions along them cannot be interpreted (data not shown). Thus, it is not possible to say whether all the details of the organization of the hydrophobin molecules at the air/water interface and on the silicon substrate are the same.

4. Discussion Both proteins, HFBI and HFBII, adsorb to the air/water interface and self-assemble there into two-dimensional, crystalline rafts. HFBI initially forms rafts of hundreds of micrometers in diameter with well-defined boundaries. The rafts consist of domains of sizes less than 2 µm. HFBII, on the other hand, covers the surface more homogeneously with the small domains. Increasing the pressure leads to coalescence of the domains into uniform and smooth films. In the nanometer length scales, the crystalline areas of the films were probed by grazing-incidence X-ray diffraction. The HFBI and HFBII films were similar in terms of the crystal structure (hexagonal with a ) 54 Å and a ) 55 Å, respectively) and thicknesses (28 Å and 24 Å). The amino acid sequence similarity between HFBI and HFBII is 69%,34 and their folded structures are very similar.8,9 This enables similar packing at the air/water interface. The HFBI and HFBII crystallites at the air/water interface were found to be stable. Repeated measurements indicated that the protein films did not suffer from radiation damage. The crystal structure of the films did not change as a function of pressure, and the films could be transferred to a solid substrate. The GID results indicate that the crystallites cover the trough surface best at 20 mN/m (HFBI) or 30 mN/m (HFBII). The BAM results confirm that at these pressures the films are the smoothest. Increasing the pressure further forces the crystallites too close to each other and leads to the beginning of collapse. The Bragg rod profiles of HFBI and HFBII were very clear and could be resolved up to q ) 0.7 1/Å. For comparison, in a recent study Miller and co-workers probed the structure of cholera toxin bound to its ganglioside receptor in a lipid monolayer at the air/water interface.31 They were able to record Bragg rods up to q ) 0.2 1/Å and attempted to model the two-dimensional protein films using the 3D single crystal structures. However, they also reported difficulties and had to settle on using tilted cylinders to describe the cholera toxin complexes. The question of why the atomic structures obtained from 3D single crystals cannot be used to describe the two-dimensional films at the air/water interface is an interesting one. The

1618 Langmuir, Vol. 25, No. 3, 2009

asymmetric units or other multimeric complexes in the single crystals cannot be used, because the multimerization of the molecules in the crystals is influenced by the exact crystallization conditions. For example, for HFBII three different crystal structures with three different asymmetric units have been reported using different salts or detergents.9,38 Therefore, instead of focusing on the supramolecular assemblies, it seems more logical to use the invidual monomers as building blocks. This assumes that the conformation of the protein molecules is the same in the single crystal and at the air/water interface. While this is not true for most of the proteins, the results from circular dichroism indicate that the secondary structures of HFBI and HFBII do not change upon adsorption and self-assembly.10 However, smaller conformational changes are possible, as, for example, in a monoclinic crystal structure of HFBII (2PL6) two types of monomers are observed. They differ only in the hydrophobic patch, which has either a closed or a more extended conformation.38 This kind of flexibility would blur the electron density and impede the interpretation. Nevertheless, a perhaps more important issue here is the effect of water and degree of hydration of the molecules. HFBI and HFBII contain a clear hydrophobic surface area on one side of the molecule. The surface is otherwise mainly hydrophilic. This, alongside the complex inner structure of the proteins, may lead to a nonuniform distribution of water molecules in and around the proteins, which would change the electron density and X-ray contrast. Combining the effects of flexibility and electron density changes might cause part of the protein molecule to be invisible to the GID experiment. That would explain why in our models using spheres the best sphere radius was only 8 Å. For both HFBI and HFBII, dimers arranged in helical rings were found to be a plausible model. Amphiphilic dimers have been previously observed in one crystal structure of HFBII.38 However, in that case both monomers of the dimer were almost on the same level, whereas here the second sphere is 20 Å above the first one. It should be emphasized that in these simplistic sphere models the maxima along the Bragg rods arise directly from the structure factor, i.e., the positions of the spheres, and are only modulated by the form factors of the spheres. Therefore, in these models the spheres have to be lifted with respect to each other to create the observed oscillations. The models do not take into account the inner structure of the proteins, which in some cases might also contribute. Nevertheless, this kind of arrangement of protein molecules would fit the AFM data of HFBI22,23 and HFBII22,38 LB films and looks different from above and below, as was observed when comparing HFBI LB and LS films.23 Note that in our model the choice of “upper” and “lower” surface is arbitrary, because turning the film upside down does not change the scattering pattern. Also, a more detailed comparison with the AFM results is difficult due to the different substrates. The dimeric building blocks used in the GID model would explain the recent observation that covalently dimerized HFBI adsorbs to the air/water interface and lowers the surface tension of water in a similar way as the native HFBI.20 It also indicates that there are specific protein-protein interactions in the lateral direction, which determine the ordering of the protein molecules in the crystalline films. The protein-protein interactions also account for the observed differences between HFBI and HFBII films and may explain the strong tendency of these proteins to self-assemble into hexagonally ordered crystallites, already in zero pressure.

Kisko et al.

In addition to HFBI and HFBII, the T. reesei genome contains at least four other hydrophobin genes.39 Of these, one protein, HFBIII, has been previously isolated, purified, and characterized at the air/water interface.30 The films formed by HFBIII were very similar to those of HFBI and HFBII in terms of the unit cell structure (hexagonal with a ) 56 Å) and stability of the structure with increasing surface pressure. These results suggest that the proteins may complement each other in the fungus. For example, the hfb2 gene is expressed in lactose-containing medium, while hfb1 is not. The observation that a mutant, from where the hfb2 gene was deleted, can still form aerial hyphae in lactose, suggests that another hydrophobin compensates for the absence of HFBII.32 Similarity of the film structures would facilitate the compensation. Hydrophobins are divided into classes I and II based on their hydropathy patterns.40 The T. reesei proteins HFBI and HFBII studied here belong to class II hydrophobins. Both the class I and class II hydrophobins are very surface-active, adsorb to the air/ water interface, and self-assemble into ordered structures. All hydrophobins may also share the same disulfide pairing,8,9,41 possibly leading to similar folded structures. According to NMR results, a class I hydrophobin, EAS from Neurospora crassa, has a β-barrel similar to HFBI8 and HFBII.9 However, EAS contains also two long, disordered loops absent from the T. reesei proteins and has a clear separation of charged and uncharged areas on its surface.41 The structural differencies are likely to account for the different morphologies of the self-assembled films of class I and class II hydrophobins, as the class I films consist of 10 nm thick rodlets. The adsorption and self-assembly processes of the class I SC3 from Schizophyllum commune also differ from those of the T. reesei hydrophobins: whereas HFBI and HFBII maintain their secondary structures upon adsorption to the air/water interface,10 the adsorption of SC3 is followed by conformational changes.21,42 Furthermore, the hexagonal structure of the selfassembled HFBI and HFBII films is obtained immediately, as against the final SC3 rodlet film is obtained only after some hours of incubation.21 This is reflected also in the lowering of surface tension of water, as HFBI and HFBII reach the minimum values within minutes (HFBI) or instantly (HFBII), while for SC3 some hours of incubation are required.10

5. Conclusions Hydrophobins are very surface-active proteins. They adsorb to the air/water interface and self-assemble into highly ordered films there. Here, we followed in situ the self-assembly of two T. reesei hydrophobins at multiple length scales. Both HFBI and HFBII formed large rafts of hundreds of micrometers in diameter. The thicknesses of the rafts were below 30 Å. The rafts contained highly crystalline, hexagonally ordered areas, which were modeled to contain six molecules in the unit cells. Hydrophobins HFBI and HFBII are rigid proteins with an amphiphilic surface. This unique structure enables the adsorption at the air/water interface without large conformational changes. At the interface, the hydrophobins self-assemble into crystalline films. The self-assembly is likely to be driven by the specific protein-protein interactions in the lateral direction. These interactions stabilize the films at the air/water interface and may (39) Neuhof, T.; Dieckman, R.; Druzhinina, I. S.; Kubicek, C. P.; NakariSeta¨la¨, T.; Penttila¨, M.; von Do¨hren, H. FEBS J. 2007, 274, 841–852. (40) Wessels, J. G. H. Annu. ReV. Phytopathol. 1994, 32, 413–437. (41) Kwan, A. H. Y.; Winefield, R. D.; Sunde, M.; Matthews, J. M.; Haverkamp, R. G.; Templeton, M. D.; Mackay, J. P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3621–3626. (42) de Vocht, M. L.; Scholtmeijer, K.; van der Vegte, E. W.; de Vries, O. M. H.; Sonveaux, N.; Wo¨sten, H. A. B.; Ruysschaert, J.-M.; Hadziioannou, G.; Wessels, J. G. H.; Robillard, G. T. Biophys. J. 1998, 74, 2059–2068.

Self-Assembled Films HFBI and HFBII

also enable the transfer of the films to solid substrates. The unusual properties of hydrophobin proteins have rendered them also as promising candidates in applications varying from coatings which modify surfaces43 or create specific adsorption cites44 to stabilization of foams and bubbles.2,11 Acknowledgment. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation (43) Scholtmeijer, K.; Jansse, M. I.; Gerssen, B.; de Vocht, M. L.; van Leeuwen, B. M.; van Kooten, T. G.; Wo¨sten, H. A. B.; Wessels, J. G. H. Appl. EnViron. Microbiol. 2002, 68, 1367–1373. (44) 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, 3021–3027.

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facilities, and we would like to thank Dr. Oleg Konovalov and Dr. Amarjeet Singh for assistance in using beamline ID10B. We thank Dr. Teemu Ikonen, Tuomas Kulomaa, and Kirsi Leppa¨nen for assistance in part of the measurements at ID10B and Mikko Suominen for the BAM measurements. Financial support from the National Graduate School in Material Physics (KK), National Graduate School in Informational and Structural Biology (GSz) and Vilho, Yrjo¨ and Kalle Va¨isa¨la¨ Foundation (KK) is gratefully acknowedged. Supporting Information Available: Brewster angle microscopy images of HFBI and HFBII at different surface pressures. This material is available free of charge via the Internet at http://pubs.acs.org. LA803252G