3290
Langmuir 1994,10, 3290-3295
Two-DimensionalProtein Array Growth in Thin Layers of Protein Solution on Aqueous Subphases Hideyuki Yoshimura,* Tschangiz Scheybani,?Wolfgang Baumeister,? and Kuniaki Nagayama Protein Array Project, ERATO, JRDC, 5-9-1 Tokodai, Tsukuba, 300-26,Japan Received March 22, 1994. In Final Form: June 14, 1994@ A simple method for making two-dimensional(2D)protein arrays using a new spreading technique was developed. A solution of the iron storage protein ferritin was injected into an aqueous subphase that had a higher density and surface tension than protein solution. The buoyancy of the protein solution made it rise to the surface where it spread quickly and smoothly. For the stage of rising and spreading, the buoyancy and surface tension were critical factors. The proteins that reached the air-water interface unfolded instantaneously and formed a continuous film. These films were transferred onto holey carbon films for transmission electron microscopic analysis. The holes of the carbon film were found to be covered with a smooth, amorphous film of unfolded protein onto which intact ferritin was adsorbed. For the stage of 2 D array formation, the presence of cadmium ion in the subphase was a critical factor. The 2D array formation occurred in the thin layer of protein solution caught between the unfolded protein film of the surface and the subphase below.
1. Introduction Ordered two-dimensional (2D)arrays of proteins are of considerable practical interest in fields as diverse as the design of nanotechnological devices' and electron crystallography.2 Perfectly flat substrates are regarded as essential for supporting the growth of 2D crystals. Therefore, the freshly cleaved surface of mica3x4or the surface of mercury5-' have been used for crystallization. Although several proteins have successfully been crystallized on mercury, this technique has the disadvantage that sophisticated equipment is required to ensure a clean surface. Recently there has been considerable success in using lipid-water interfaces for 2D c r y s t a l l i ~ a t i o nThe .~~~ design of lipid polar head groups that interact with proteins is the key point of this method. Creating a suitable charge density is sufficient for some systems, while others require that specific ligands be attached to the lipid polar head groups.2 In addition to recent work on 2 D crystallization, the surface chemistry of protein spreading and protein monolayer formation at the air-water interface has been the subject of extensive investigations.10-21 To achieve com-
* To whom correspondence should be addressed. t Max-Planck-Institut fur Biochemie, Martinsried, D82152, Germany. Abstract published inAdvance ACSAbstracts, August 15,1994. (1)h m , D.; S i r a , M.; Messner, P.; Sleytr, U. B. Nanotechnology 1991, 1, 5. (2) Jap, B. K.; Zulauf, M.; Scheybani, T.; Hefti, A.; Baumeister, W.; Aebi, U.;Engel, A. Ultramicroscopy 1992,46,45. (3)Horne, R. W.; Pasquali-Ronchetti, I. J.Ultrastruct.Res. 1974,47, 361. (4)Harris, J. R. Micron Micros. Acta 1991,22,341. (5)Yoshimura, H.; Matsumoto, M.; Endo, S.; Nagayama, K. Ultramicroscopy 1990,32,265. (6)Ishii, N.; Taguchi, H.; Yoshida, M.; Yoshimura, H.; Nagayama, K. J . Biochem. 1991,110,905. (7)Ishii, N.; Yoshimura, H.; Nagayama, K.; Yoshida, M. J.Biochem. 1993,113,245. (8)Fromherz, P.Nature 1971,231,267. (9)Uzgiris, E. E.;Komberg, R. D. Nature 1983,301,125. (10)Bull, H. B. Adu. Protein Chem. 1947,3,95. (11)Yamashita,T.;Bull,H.B.J. ColZoidlnterfaceSci.1967,24,310. (12)Stlllberg, S.;Teorell, T. Trans. Faraday SOC.1939,35, 1413. (13)Dervichian, D.Nature 1939,144,629. (14)Bull, H. B.J.B i d . Chem. 1950,185,27. (15)Cheesman, D. F.;Schuller, H. J. Colloid Sci. 1954,9, 113. (16)Augenstine, L. G.;Ghiron, C. A.; Nims, L. F. J. Phys. Chem. 1958,62,1231. (17)MacRitche, F.;Alexander, A. E. J. Colloid Sci. 1963,18,458. @
plete spreading of the proteins, concentrated salt solutions that have a higher surface tension than water ( e g . ,3 M N&S04) have been used as the subphase.'0J1J6 Alternatively, alcohols such as isopropyl alcohol,which decrease the surface tension, were added to the protein solution to promote spreading.12J3 It was reported that unfolded protein films were formed a t the air-water interface when protein solutions were spread on a n aqueous s u b p h a ~ e . ' ~ - ' ~ The smallest occupied area of the unfolded protein film was measured and found to be about 0.78 m2/mgof protein (0.15nm2/aminoacid residue).1° At this area density, the air-water interface is presumably covered completely with a monolayer of the unfolded protein film. When a larger amount ofprotein than in the former condition was spread, the behavior of the n-A isotherm (surface pressure versus occupied area per molecule) changed from the behavior that is normally observed for monolayer coverage. Here the enzymatic activity of spread protein (pepsin,15 trypsin,16J8 acetylcholinesterase,19 and catalasez0) was conserved. Many molecular models were proposed to explain this phenomena. Cheesman and Schuller15proposed the adsorption of intact proteins on the unfolded protein film, while Augenstine et a1.I8 suggested that intact protein was incorporated into the unfolded protein film. There have been many discussions regarding the structure of these films, but these have not led to a unanimously accepted conclusion. This discussion reflect the lack of information about the real structure of such films, which can only be provided by direct imaging techniques such as microscopy. In this communication we describe a transmission electron microscope (TEM) investigation of protein films obtained with a new, simple spreading technique that involves injection of the protein solution into the subphase (followed by smooth rising) rather than dropping the protein solution on the subphase (which disrupts the surface). This technique takes advantage of the buoyant force and the surface tension of protein solutions, which are important factors when the solutions and the subphase have different densities and surface tensions. The proteins form a thin, smooth monolayer of unfolded protein a t the air-water interface. Two-dimensional protein arrays are (18)Augenstine, L. G.;Ray, B. R. J.Phys. Chem. 1957,61, 1385. (19)Skou, J. C. Biochim. Biophys. Acta 1959,31, 1. (20) Nitsch, W.; Maksymiw, R. Colloid Polym. Sci. 1990,268,452. (21)MacRitche, F.Anal. Chim. Acta 1991,249,241.
0743-7463/94/2410-3290$04.50/0 0 1994 American Chemical Society
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Two-Dimensional Protein Array Growth obtained when enough intact protein is available to adsorb to this film. To promote the growth of the array in two dimensions, but not in three dimensions, a thin protein solution sandwiched between the smooth unfolded protein film and the dense aqueous subphase is considered to be crucial.
2. Experimental Section 2.1 Materials. Horse spleen holoferritin (with iron core)was purchased from Boehringer Mannheim and was used without further purification. Recombinant apoferritin (withoutiron core), for which the DNA sequence was cloned from horse liver, was expressed in E-Coli22and purified as described before.23 Recombinant Thermoplasma acidophilum proteasomes were isolated as described before.24 The chemical reagents (glucose (Merck), glycerol (Merck), dimethyl sulfoxide (Sigma), poly(ethyleneglycol)6000 (Fluka),MOPS(Merck),and MES(Dojind0)) were of the highest obtainable purity. The water used was distilled twice or purified by use of a Millipore filter apparatus. 2.2 Trough and Surface Tension Measurements. A homemade circular Teflon trough (15 mm diameter, 177 mm2 surface area)was filled with an aqueous subphase (0.5mL). The protein solutions (0.8-5 pL) were injected into the subphase with a Hamilton syringe (No. 701) using the method described below. The surface tension was measured at room temperature using a Wilhelmyplate (2 x 15mm2 filter paper) with a tension balance (Riegler& Kirstein, Wiesbaden). The accuracywas f O . l mN/m. 2.3 ElectronMicroscopy. The TEM grids were covered with either a continuous or a holey carbon film. The holey carbon grids were prepared accordingtothe method describedby Reichlet et al.25 The film at the air-water interface was transferred by touching the grid horizontally (face to face) to the surface. Electron micrographs were taken under low electron dose conditionsusing either a Philips CMlO or JEOL JEM1200EX-I1 electron microscope (the total electron dose was less than 2000 e-/nm2). Negatives were digitized by use ofa CCD camera system (Eikonix CCD camera 1412) or a microdensitometer (PerkinElmer Model 1010M). For image processing,the SEMPER and EMIDO (developed by this ERATO project) software systems were used. 2.4 Spreading Method. The spreading of the protein solutions on water surfaces is usually done by dropping the solution onto the surface. With this method, the water surface is disturbed by surfacevibration,resulting in poor reproducibility. To obtain better spreading, we injected the protein solution directly into the subphase solution. With the proper density and surface tension, the protein solution rises to the surface and spreads without any disturbance at the surface. This process was easily visible when using a holoferritin solution, which has a red color due to the iron core. 2.6 Aqueous Subphases. For testing the parameters that affect the spreading, modified aqueous subphases containing glucose(2,4,10,and 20%(w/v)),NaCl(0.5M),dimethyl sulfoxide (loo%),poly(ethy1eneglycol) (10%(w/v)),and glycol (10%(v/v)) solutions were used. The densities and the surface tensions of these solutions are summarized in Table 1. For optimum 2D array formation experiments, the aqueous subphase contained 2% glucose, 0.15 M NaCl, 10 mM CdS04, and 10 mM'MOPS (or MES) (pH 5.7). 3. Results 3.1 Aqueous Subphases. Different aqueous subphases including glucose, NaC1, dimethyl sulfoxide (DMSO), poly(ethy1ene glycol) (PEG SOOO), and glycerol were tested to observe their efficiency in promoting (22) Takeda,S.;Ohta,M.; Ebina, S.;Nagayama,K.Biochim.Biophys. Acta 1993, 1174, 218. (23) Banvard, S. H.; Stammers, D. K; Harrison, P. M.Nuture 1978, 271, 282. (24) Piihler, G.;Weinkauf, S.; Bachmann, L.; Miiller, S.;Engel, A.; Hegerl, R.; Baumeister, W. EMBO J. 1992,11, 1607. (25) Reichelt, R.; Konig, T.; Wangermann, G. Micron 1977, 8, 29. (26) Described in the Sigma catalog. (27) Our measurement at room temperature. (28) CRC Handbook of Chemistry and Physics, 72th ed.; CRC Press: Boca Raton, FL, 1991.
Table 1. Surface Tension and Density of Solutions Used for the Aqueous Subphase surface temp tension - y density spreadconc ("C) y(mN/m) (mN/m) (g/cmS) ing~' water28 100% 18 73.05 0 0.9985 100% 20 72.75 0 0.9982 glucose27 2% (w/v) -20 75.6 -2.8 1.02 0.5 M 20 73.8 -1.0 1.02 NaC128 100% -20 52.0 +20.8 1.09326 + ~ ~ ~ 6 0 0 0io%(w/v) 27 66.0 +6.8 1.1 -20 $0.2 1.03 glycerol28 10%(v/v) 18 72.9
+ +
"+"
=For spreading, indicates complete spreading and L'' indicates that the spread protein solution remained as a disk.
1 4
4,'
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"0
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Figure 1. Spreading of holoferritin on aqueous subphases. Holoferritin (5mg/mL, 0.8pL) was spread on aqueoussubphases containing 2% glucose (0)and 10%glycerol (A).The diameter of the circular disk of protein solution spread on the subphases was measuredfromthe video recording. On the glucoseaqueous subphase,the holoferritin solutionspread over the entire surface area within 1s. On the glycerol aqueous subphase, the protein solution remained as a small circular disk for periods longer than 30 s.
spreading. The spreading of holoferritin solutions (5 mg/ mL, 0.8,uL)was monitored with a CCD camera or observed by eye. Complete concentric spreading was obtained with glucose- and NaC1-containing subphases. On a DMSO subphase (100%DMSO), the holoferritin solution spread in a spokelike manner rather than concentrically. For subphases containing PEG and glycerol, the holoferritin solution rose to the surface and formed a circular disk that spread only slowly. The time courses of the spreading in the presence of glucose and glycerol subphase are shown in Figure 1. On the glucose-containing subphase, the ferritin solution spread within 1 s. In contrast, on the glycerol-containing subphase the solution remained as a circular disk (6 mm diameter) for u p to 30 s after it rose to the surface (Figure 1). A higher surface tension than water (Table 1)is considered to be important for fast spreading. Subphaseswith smaller surface tensions, such as glycerol and PEG, yielded incompletespreading (Table 1). Although DMSO has a smaller surface tension than water, the holoferritin solution spread well though not concentrically. Thus, the spreading mechanism may involve other physicochemical properties instead of or in addition to the surface tension. The film at the air-water interface was transferred to TEM grids covered with a carbon support film. When the subphase was DMSO or a concentrated glucose solution (higher than lo%), the transferred film had to be rinsed
3292 Langmuir, Vol. 10,No.9,1994
Yoshimura et al.
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Figure 2. Effect of ions on the packing of holoferritin. Images of the transferred holoferritin film (without negative staining) were digitized and the Fourier transforms calculated. Panel A is the image of the holoferritin film transferred from the aqueous subphase (2% glucose, 0.15 M NaC1, and 10 mM CdS04). The radial distribution function (B) was obtained from rotational averaging of the power spectrum of the Fourier transform of the image (inset of B). The position of the first diffraction ring and the line width of the peak from the radial distribution functions were estimated as a measurement of the packing of the holoferritin molecules. These values are plotted in part C for different salts (10 mM of CdS04 without NaCl (O), 10 mM of CdS04 (W), CdC12 (A), MgS04 (e), MgC12 (O),ZnCl2(+), and MnC12 ( x 1)in the subphase (2%glucose and 0.15 MNaC1). The pH ofthe solutions was 6.0 f 0.5. Adjusting the pH to 5.7 with 10 mM MOPS buffer (in 2% glucose, 0.15 M NaC1, and 10 mM of CdS04) improved the reproducibility of the packing density (Le., position of the peak) (A). The transferred holoferritin film showed the sharpest line width when spread on a 2% glucose aqueous subphase with 10 mM CdS04,0.15 M NaCl, and 10 mM MOPS (pH 5.7). The holoferritin molecules packed in a well-ordered array under these conditions.
with water to produce clear negative strain images. Small assemblies of holoferritin arrays with hexagonal lattices were observed when the subphase was glucose and NaC1. A 2% glucose solution was used as the aqueous subphase in the following experiments. 3.2 Salt Conditions in the Subphase. It is known that the ions in protein solutions have an influence on protein-protein interactions. For successful crystallization of protein molecules, the salt conditions must therefore be optimized.2 To investigate the effect of salt ions on the ordering of holoferritin, various salts were used, and the resulting arrays were examined by TEM by transferring them to a carbon film. Electron micrographs with similar focus settings were digitized and their Fourier transforms calculated. The radial distribution functions of the power spectrums were obtainedby averagingthe power spectrum rotationally (Figure 2B). The position and the half-width of the peak of the radial distribution functions were measured as a value of the packing state. In addition to the 2% aqueousglucose solution,NaCl(O.15M) was always included in the aqueous subphase to improve the reproducibility and uniformity of the film. Figure 2C shows the line widths and the positions of the peak in the radial distribution functions obtained for the various salts examined (CdS04, CdC12, MgS04, MgC12, ZnCl2, and MnC12). The line width is a criterion of the quality of the arrays;
well-ordered arrays give sharper line widths and poorly ordered arrays give broader ones. The position of the peak is related to the averaged distance between molecules. The position of the peak usually correlates with the line width (Figure 2C) because closer molecular contacts tend to improve the quality of the lattice. The narrowest line width was observed with 10 mM CdS04 (Figure 2C). Therefore, we selected the aqueous subphase with 2% glucose, 0.15 M NaC1, and 10 mM CdS04 for further crystallization experiments. In the initial experiments unbuffered solutions were used (pH about 6.0'f 0.5). The later experimentsincluded 10mMMOPS or MES to adjust the pH of the solution to 5.7. 3.3 Unfolding of Protein at the Air-Water Interface. Holoferritin solution (1mg/mL, 1pL)was spread on the subphase (2%glucose, 0.15 M NaC1,lO mM CdS04, and 10 mM MOPS (pH 5.7)), and the film formed a t the air-water interface was transferred to a holey carbon film 5 min after spreading. Due to the iron core of holoferritin, it can easily be seen by TEM without staining. The holoferritin film covered most of the holes of the carbon film (Figure 3A), though it was highly susceptible to damage by electron irradiation (Figure 3B). To avoid this, the protein film was reinforced by a carbon coating.2The film was rinsed with water from the other side to remove excess glucose. A typical electron micrograph is shown in Figure 3C. The presence of apparently isolated ferritin
Two-DimensionalProtein Array Growth
Langmuir, Vol. 10, No. 9, 1994 3293
Figure 3. Electron micrographs of holoferritin film transferred to holey carbon films. The holoferritin film was transferred from the surface of the interface to a holey carbon film. The excess glucose solutionon the grid was removed by blotting with filter paper. The specimen was then rinsed with water (A and B) and observed by electron microscope without staining. The transferred film was coated with carbon (3-5 nm) from the back side (i.e., the surface directed up toward the air above the trough) (C). The holes in the holey carbon film were covered with a holoferritin film (A). These films were often disrupted during electron irradiation (B). The carbon coating reinforced and stabilized the holoferritin film (C). The existence of the isolated holoferritin molecules on the hole of the grid suggests that a thin amorphus film, probably formed by unfolded protein, supports these molecules (see the circled area in the magnified image; inset of C).
A
C
Figure 4. Absorption of intact apoferritin onto the unfolded protein film. The recombinant apoferritin film was transferred to a holey carbon film 1 min (A) and 3 min (B)after the protein was spread. The unfolded protein films were reinforced by carbon coating and negatlivelystained with 2% uranyl acetate. In image A, unfolded protein films with adsorbed apoferritin (a), ruptured unfolded protein films (b), and vacant holes (c) were observed. Nucleated hexagonal arrays are shown in image B with an inset of the magnified image.
molecules across the hole (inset of Figure 3C) confirms that these are supported by a thin amorphous film. This film is considered to be unfolded protein because no film could be detected for subphases that had not been injected with protein solution. 3.4 Growth of Two-Dimensional Protein Arrays under the Unfolded Protein Films. To investigate the time course of formation of the unfolded protein film, recombinant apoferritin (1 mg/mL, 1pL)was spread on the same subphase as described in section 3.3,and samples were taken after different time periods. The films were
transferred to holey carbon films and negatively stained with 2% uranyl acetate after carbon reinforcement. One minute afterthe protein was spread, the holes of the carbon film were already observed to be covered with a smooth unfolded protein film (Figure 4A). A few intact apoferritin molecules that were adsorbed onto this film were also observed. Three minutes after the protein was spread, the number of adsorbed apoferritin molecules increased, and small hexagonal arrays of apoferritin were observed (Figure 4B). Apoferritin formed large domains of hexagonal arrays 10min after the protein was spread (Figure
3294 Langmuir, Vol. 10, No. 9, 1994
Figure 5. Two-dimensional array of apoferritin. The recombinant apoferritin film was transferred to a holey carbon film 10 min after the protein was spread and processed as in Figure 4. The adsorbed apoferritin formed a large 2D array. The inset is the power spectrum of the Fourier transform for the center area (560 x 560 nm2). The diffraction spots extend to the sixth order (the signal to noise ratio (S/N) is 3.6 for the spot (6,O)). This array is found to be a well-ordered crystal by displacement field analysis (DFA).
5). These observations suggest that the unfolding of protein molecules took place first and subsequently intact protein molecules adsorbed on the film. The 2D arrays were also observed after transfer to continuous carbon support films. Compared with the holey carbon films, however, the frequency with which ordered arrays were successfully observed was much reduced. The unfolded protein films may have tended to dissociate from the carbon support film during the staining process. The carbon coating used to reinforce the unfolded protein films that were transferred to the holey carbon films would fix the film itself as well as the molecules adsorbed on the film. When the total amount of protein that was spread on the surface was reduced to 0.5pg (0.5 mg/mL, 1pL),a film of unfolded protein with a small number of intact spherical molecules adsorbed onto it was observed. From this, we can conclude that the total amount of protein required to cover the surface of the trough (177 mm2)in the form of an unfolded protein film was about 0.5 pg. 3.5 Crystallinity of 2D Arrays. To examine the crystallinity of two-dimensional arrays of apoferritin, correlation averaging of the TEM image^^^.^^ followed by displacement field analysis (DFA)a1>32 were accomplished. The cross-correlation function between a small reference image extracted from the window-filtered image of the array and the original image of the array was calculated to determine the displacements of unit cells from the ideal lattice More than 4500 correlation peaks (unit cells sites) with little displacementwere found in the array (Figure 5 ) , and the averaged image aRer compensation for the displacement of unit cells (correlation averaging) was calculated. As we reported b e f ~ r e ,the ~ averaged (29)Saxton, W.0.; Baumeister, W. J . Micros. 1982,127,127. (30)Frank, J. Optik 1982,63, 67. (31)Dum, R. Ultramicroscopy 1991,38,135. Dum, R.; Baumeister, W. Ultramicroscopy 1992, (32)Saxton, W.0.; 46, 287.
Yoshimura et al. image showed threefold symmetry, and the molecules are considered to be oriented with their threefold axes perpendicular to the crystal plane. Because the lattice displacements also exhibit local distortions of unit cells, the distortion (deformation and rotation) of the unit cells in the array was analyzed by DFA. The histograms of the deformation (magnification and elongation) and rotation that were obtained showed Gaussian distribution, and the calculated standard deviations (a)of magnification, elongation, and rotation were 0.60%, 0.42% (against the first principal axes), and 0.30°, respectively. These small standard deviations reveal that the array is a crystal with only slight distortions. Further details of the structure analysis of the apoferritin 2D crystal will be published elsewhere. 3.6 Unfolded Protein Film at the Surface of Bulk Protein Solutions. Ordered 2D arrays (crystals) of apoferritin were obtained by spreading a protein solution on the surface of an aqueous subphase. The growth of 2D arrays may, however, also be able to be done a t the surface of bulk protein solutions without the need for our injection and spreading procedure. To examine this alternate method, protein films were transferred from the surface of bulk apoferritin solutions that had protein concentrations ranging from 0.003 to 0.9 mg/mL and the same type of solution conditions as the subphase (2% glucose, 0.15 M NaC1, and 10 mM MES (pH 5.7)) used for spreading experiments. In the presence of 10 mM CdS04, the apofemtin solutionsbecame turbid (CdS04had previously been used in 3D crystallization experiments for protein pre~ipitation~~). Unordered aggregates as well as thick 3D crystals were adsorbed onto the unfolded protein film, but no 2D arrays were found in this protein concentration range. In the absence of CdS04, only random adsorption of apoferritin molecules to the unfolded protein film was observed. 3.7 Surface Pressure of Spread Apoferritin and the Bulk Apoferritin Solution. The surface pressure of apoferritin (1 mg/mL, 1 pL) spread on the aqueous subphase increased up to 2.8 mN/m and was found to reach a stable value 20 min after the protein was spread (Figure 6). No detectable surface pressure was observed when the total amount of spread protein was less than 0.5 pg. Also, no surface pressure was detected for the bulk apoferritin solution under the same solution conditions as those used in the spreading experiments. In the absence of CdS04, the surface pressure increased gradually (Figure 6). 3.8 Application of This Spreading Technique to Other Proteins. We applied this technique to proteasome (a multicatalytic proteinase) using the same subphase conditions as used for ferritin (2% glucose, 0.15 M NaC1,lO mM CdS04, and 10 mM MOPS (pH 5.7)). A 1 pL protein solution with a concentration of 0.15 mg/mL was injected five times to supply enough protein molecules to form both the unfolded protein film and adsorbed intact molecules below that. The proteasomes were found to also form an unfolded protein film to which small hexagonal arrays comprised of 40-50 adsorbed molecules were observed (Figure 7).
4. Discussion 4.1 Unfolded Protein Film at the Air-Water Interface. Accordingto Bull,lothe smallest occupied area of unfolded protein is 0.78 m2/mg of protein, independent of the species of protein. With this value for our system, 0.23 pg of protein is required to completely cover the surface of the 177 mm2 trough. When we spread 1pg of apoferritin, 23% of the total protein should cover the water-air interface as unfolded protein film, and 77% of the total protein should remain intact. Because the
Langmuir, Vol. 10, No. 9, 1994 3295
Two-Dimensional Protein Array Growth
limit of 0.2 mN/m) because no significant increase in surface pressure was detected when the amount of the spread protein was increased up to 0.5 pg (the point a t which unfolded protein film formation is complete).
8 -
4.3 Adsorption of ApoferritinMolecules onto the Unfolded Protein Film. A gradual increase and then leveling off of the surface pressure after the protein was spread was observed (Figure 6). This effect can be explained as being due to the adsorption of intact apoferritin molecules to the unfolded protein film (Figure 4). The gradual increase in the surface pressure of the
n
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Figure 6. Surface pressure of apoferritin film. The surface pressure of apoferritin (1mg/mL, 1pL)spread on the subphase (2% glucose, 0.15 M NaC1,lO mM CdSO4, and 10 mM MOPS (pH 5.7)) is measured by means of the Wilhelmy method. The different symbols (A,.) correspond to two independent experiments carried out under the same conditions. The surface pressure of the bulk apoferritin solution (1 mg/mL) was not detected under the same solution conditions as those used in the spreadingexperiments. In the absence of CdS04,the surface pressure of the bulk apoferritin solution increased gradually (40).
Figure 7. Electron micrograph of proteasomes arrays. Proteasomes from 5". acidophilum were spread on the glucose aqueous subphase with the same procedure and conditions as those used for ferritin. Small 2D arrays were observed.
smallest occupied area of an intact spherical ferritin molecule is 133 nm2 (0.18 m2/mg protein), the rest of the spread protein (0.77 pg) can cover 79% of the area under the unfolded protein film. This rough estimation well explains the observed density of adsorbed apoferritin molecules on the film. Because almost all of the protein is incorporated in either the unfolded protein film or the adsorbed molecules, the amount of protein that diffises into the subphase appears to be negligible. 4.2 Surface Pressure of the Unfolded Protein Films. The surface pressure due to the unfolded film was estimated to be very small (less than the instrumental
bulk protein solution occurred because protein molecules can be supplied from the bulk phase without limitation. The leveling off of the surface pressure is considered to be due to the limited supply of protein molecules from the thin solution layer on the aqueous subphase. In the presence of CdS04, the surface pressure of the bulk apoferritin solution was below the limits of measurement. Under these conditions,apoferritin molecules aggregated and the solution became turbid. Adsorption of isolated solid aggregates to the unfolded protein film does not appear to contribute to the surface pressure because of lack of interaction between the aggregates. Furuno et aZ.33reported the adsorption of ferritin to a positively charged polypeptide(poly(1-benzyl-L-histidine)) layer a t the air-water interface. Because the isoelectric point of ferritin is about pH 4.8,34 resulting in ferritin molecules having a negative charge at neutral pH, ferritin is able to bind to a positively charged layer. Though the unfolded ferritin film is consideredto have the same charge as the intact ferritin molecule (negativecharge at pH 5.7), the unfolded protein has both positively and negatively charged amino acids distributedthroughout the structure. In our experiments, intact ferritin likely binds to the positively charged sites of the unfolded polypeptide chains. 4.4 Growth of 2D Arrays. We observed 2D protein arrays only when protein solutions were spread underneath the air-water interface. The formation of a thin protein solution layer on the aqueous subphase appears to confine the growth of the array in two-dimensions. According to Bull,lothe thickness of the unfolded protein film is about 1 nm, which corresponds roughly to the thickness of a polypeptide chain. This thin and flat film (on a nanometer scale) appears to provide an ideal substrate for 2D crystallization. We obtained small hexagonal arrays of the proteasomes on the unfolded protein film, but the conditions need to be further optimized to form better crystals that can be analyzed by electroncrystallography. Because it is generally expected that proteins will unfold at the air-water interface and form mono1ayers,l0-l8our technique should work for many other water-soluble proteins.
Acknowledgment. We are grateful to Dr. Reiner Hegerl of the Max-Planck-Institute for helpM discussions and for help on image analysis using the SEMPER system. We are also grateful to Dr. Dieter Typke for his advice on the transmission electron microscopymeasurements. The recombinant apoferritin was supplied by Dr. Shigeki Takeda of the Protein Array Project, and the recombinant proteasomes were supplied by Mr. Peter Zwickl and Mr. Thorsten Wenzel of the Max-Planck-Institute. We also thank Dr. Gills Picard of the Protein Array Project for his critical reading of this manuscript. (33) F'uruno, T.; Sasabe, H.; Ulmer, K. M. Thin Solid Films 1989, 180, 23.
(34)Urushizaki, I.; Niitsu,Y.; Ishitani, K.; Matsuda, M.; Fukuda, M. Bwchim. Bwphys. Acta 1971,243,187.
