Two-Dimensional Crystallization of Streptavidin Studied by

Brewster angle microscopy (BAM) is extended to probe protein adsorption and aggregation processes at interfaces quantitatively. Since no p-polarized l...
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Two-Dimensional Crystallization of Streptavidin Studied by Quantitative Brewster Angle Microscopy Wolfgang Frey, William R. Schief, Jr., and Viola Vogel* Center for Bioengineering, Box 357962, University of Washington, Seattle, Washington 98195 Received July 3, 1995. In Final Form: October 26, 1995X Brewster angle microscopy (BAM) is extended to probe protein adsorption and aggregation processes at interfaces quantitatively. Since no p-polarized light is reflected from the plain air-water interface under Brewster’s angle, the grayscales of BAM images solely depend on the optical thicknesses of interfacial layers; photolabeling is not required. Effective refractive indices are calculated from the grayscales via Fresnel’s equations and are finally converted to relative protein surface densities by the Maxwell-Garnett theory. It is shown here that the Maxwell-Garnett theory provides an accurate framework for the description of a protein-water slab. Streptavidin binding to biotinylated monolayers at the air-water interface was chosen as a model system for an in situ study of the formation of two-dimensional streptavidin crystals. H-shaped aggregates are seen with BAM. We identify these as two-dimensional crystals since they are equal in size and shape to those typically observed in fluorescence microscopy which have been characterized by electron diffraction. The difference in protein surface density between the streptavidin crystals and the noncrystalline surrounding is sufficient to provide for a rich contrast without the use of a fluorescence probe. A critical protein surface density equal to 75% of the crystal density is found to be required for the crystals to form, a value that is independent of the protein bulk concentration. We have studied further the compressibility of the two phases. Whereas the density of the crystalline phase remains constant during compression, the noncrystalline phase can be compressed to a surface density which exceeds that of the crystalline phase without initiating further crystal growth. This leads to an inverted contrast in BAM; dark crystals are seen on a bright background. The protein density of the noncrystalline phase can also exceed that of the crystalline phase upon the much slower process of protein adsorption from sufficiently concentrated bulk solutions. The nature of this two-dimensional phase transition is discussed.

Introduction Functionalized surfaces that are highly ordered on a molecular scale have become important tools in molecular biotechnology. They also serve as models to better understand the lateral self-organization of biological molecules as well as their interaction with biological surfaces.1,2 One approach to address the molecules of interest to interfaces is via high-affinity ligand binding.3,4 The ligands must be well anchored to the surface yet easily accesible from the bulk. One extensively studied model system is a surface layer functionalized with biotin as the ligand. Biotin binds to the protein streptavidin with extraordinary strength (Ka ) 1015 M-1). This strong recognition process between streptavidin and the vitamin biotin5 has been widely used for biotechnological applications in solution.6 The bacterial streptavidin produced by streptomyces avidinii is a 4 × 15 kDa tetramer with 159 amino acids for each monomer. It can be processed on both ends to form “core” streptavidin which has been crystallized in three dimensions. The tertiary structure of biotin bound streptavidin as well as some structural changes induced by biotin binding are resolved.7-9 Strepta* To whom correspondence should be sent ([email protected]). X Abstract published in Advance ACS Abstracts, February 1, 1996. (1) Swalen, J. D.; Allara, J.; Andrade, J. D.; Chandross, E. A.; Garoff, S.; Israelachvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932. (2) Edwards, A. M.; Darst, S. A.; Feaver, W. J.; Thompson, N. E.; Burgess, R. R.; Kornberg, R. D. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2122. (3) Uzgiris, E. E.; Kornberg, R. D. Nature 1983, 301, 125. (4) Egger, M.; Heyn, S. P.; Gaub, H. E. Biochem. Biophys. Acta 1992, 1104, 45. (5) Green, N. M. In Advances in Protein Chemistry; Anson, M. L., Edsell, J. T., Eds.; Academic Press: New York, 1975; p 85. (6) Wilchek, M.; Bayer, E. A. In Avidin-Biotin Technology; Abelson, J. N., Simon, M. I., Eds.; Methods in Enzymology; Academic Press, Inc.: New York, 1989; Vol. 184.

vidin has been further two-dimensionally crystallized at the air-water interface, and the transferred samples have been structurally characterized with lesser resolution.10 H-shaped streptavidin crystals can be visualized at the air-water interface if the protein is labeled with FITC; such crystals give rise to polarized fluorescence.11,12 Investigations with neutron and X-ray reflection at the air-water interface have given further insight into the lipid protein interaction, the role of the spacer between the lipid and the biotin,13-15 and the hydration of the protein inside the crystal.16-18 In contrast to the air-water interface, lattice matching and steric hindrance between adjacent biotins play a very important role if biotinylated amphiphiles are immobilized at a solid-water interface.19,20 Optimal binding at the solid-water interface is accomplished only if a fraction of (7) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243 (4887), 85. (8) Weber, P. C.; Wendoloski, J. J.; Pantoliano, M. W.; Salemme, F. R. J. Am. Chem. Soc. 1992, 114, 3197. (9) Pa¨hler, A.; Hendrickson, W. A.; Gawinowicz Kolks, M. A.; Argarana, C. E.; Cantor, C. R. J. Biol. Chem. 1987, 262 (29), 13933. (10) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.; Blankenburg, R.; Ribi, H. O.; Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991, 59 (2), 387. (11) Ahlers, M.; Blankenburg, R.; Grainger, D. W.; Meller, P.; Ringsdorf, H.; Salesse, C. Thin Solid Films 1989, 180, 93-9. (12) Blankenburg, R.; Meller, P.; Ringsdorf, H.; Salesse, C. Biochemistry 1989, 28, 8214. (13) Vaknin, D.; Als-Nielsen, J.; Piepenstock, M.; Lo¨sche, M. Biophys. J. 1991, 60 (6), 1545. (14) Lo¨sche, M.; Piepenstock, M.; Diederich, A.; Gru¨newald, T.; Kjaer, K.; Vaknin, D. Biophys. J. 1993, 65 (5), 2160. (15) Schmidt, A.; Spinke, J.; Bayerl, T.; Sackmann, E.; Knoll, W. Biophys. J. 1992, 63 (5), 1385. (16) Lo¨sche, M.; Erdelen, C.; Rump, E.; Ringsdorf, H.; Kjaer, K.; Vaknin, D. Thin Solid Films 1994, 242 (1-2), 112. (17) Haas, H.; Mo¨hwald, H. Colloids Surf., B 1993, 1 (3), 139. (18) Haas, H.; Mo¨hwald, H. Mater. Sci. Eng., C 1994, C1 (2), 75. (19) Ha¨ussling, L.; Ringsdorf, H.; Schmitt, F. J.; Knoll, W. Langmuir 1991, 7, 1837. (20) Spinke, J.; Liley, M.; Schmitt, F.-J.; Guder, H. J.; Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99 (9), 7012.

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the surface is functionalized with biotin. An increase in layer thickness on binding of streptavidin to biotinylated thiols on gold can be observed with surface plasmon spectroscopy and microscopy,15,21 but no regularly shaped aggregates indicating crystalline ordering have been found. In situ imaging of streptavidin crystals at the air-water interface has been limited so far to fluorescence-labeled proteins. The recent introduction of Brewster angle microscopy (BAM) to the investigation of molecular monolayers at the air-water interface22-25 has opened the possibility of imaging molecular monolayers without employing a fluorescence probe. BAM is based on Fresnel’s principles for polarized light reflected from an interface; any surface layer with a refractive index different from the adjacent media changes the intensity and polarization of the light which is reflected from the interface. The background intensity is minimized by choosing the angle of incidence to be the Brewster angle of the bare interface, at which no p-polarized light is reflected. Compared to ellipsometry, which minimizes intensities at two polarizer angles, BAM requires absolute intensity measurements, which slightly reduce the sensitivity to the index of refraction or layer thickness. At the air-water interface ellipsometry can determine the optical thickness absolutely.26 BAM is restricted to relative measurements to achieve similar accuracy although we found that absolute intensity calibrations may be possible. On the other hand, the ellipsometric imaging of dynamic behavior at an interface is very limited, and the interpretation is labor intensive.27 This is overcome in BAM by restricting the measurement to only one parameter (intensity at p-polarization) instead of two (polarizer positions), which makes spatially resolved dynamic observation possible. Introduction of an analyzer in the reflected beam of the BAM, however, has been useful in observing optical anisotropy.28 Quantitative BAM requires additional information which can be provided by ellipsometry as in the studies of streptavidin binding at the air-water interface to a partially26 and a fully29 biotinylated lipid monolayer. Although BAM has been used very successfully to investigate lipid, fatty acid, and liquid crystal phase transitions,30-32 no study of a lipidprotein system has been reported to our knowledge yet. In this paper, we quantitatively analyze protein adsorption and crystallization processes at the air-water interface with BAM. In the first part, streptavidin crystals as observed with BAM are described and compared to those crystals found by others using fluorescence-labeled streptavidin. In the second part, our approach to a quantitative image analysis is outlined from which the refractive index and the surface density of the protein layer are obtained. The adsorption of the protein to the interface is studied for different protein bulk concentrations. Two different surface-bound protein phases can be distinguished: a crystalline and a noncrystalline phase. The noncrystalline phase can eventually reach higher protein densities than the crystalline phase. In the third part we show that the two different phases exhibit different (21) Schmitt, F.-J.; Knoll, W. Biophys. J. 1991, 60 (3), 716. (22) Henon, S.; Meunier, J. Rev. Sci. Instrum. 1991, 62 (4), 936. (23) Henon, S.; Meunier, J. Thin Solid Films 1993, 234 (1-2), 471. (24) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (25) Ho¨nig, D.; Mo¨bius, D. Thin Solid Films 1992, 210/211, 64. (26) Reiter, R.; Motschmann, H.; Knoll, W. Langmuir 1993, 9, 2430. (27) Erman, M.; Theeten, J. B. J. Appl. Phys. 1986, 60 (3), 859. (28) Overbeck, G.-A.; Ho¨nig, D.; Mo¨bius, D. Thin Solid Films 1994, 242 (1-2), 213. (29) Herron, J. N.; Mueller, W.; Paudler, M.; Riegler, H.; Ringsdorf, H.; Suci, P. A. Langmuir 1992, 8, 1413. (30) Henon, S.; Meunier, J. J. Chem. Phys. 1993, 98 (11), 9148. (31) Ho¨nig, D.; Overbeck, G. A.; Mo¨bius, D. Adv. Mater. 1992, 4 (6), 419. (32) Fischer, B.; Tsao, M. W.; Ruiz-Garcia, J.; Fischer, T. M.; Schwartz, D. K.; Knobler, C. M. J. Phys. Chem. 1994, 98, 7430.

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Figure 1. Brewster angle microscopy (BAM) visualizes molecular monolayers at interfaces under Brewster’s angle. Whereas no p-polarized light is reflected from the plain airwater interface, a disturbance of Brewster’s condition, for instance by the presence of a lipid monolayer, gives rise to reflection of p-polarized light which is imaged by a lens system onto a CCD camera. The extra long working distance of our setup enables the combination of Brewster angle microscopy with other techniques, including fluorescence microscopy.

compressibilities. Comparison of the results allows us to draw conclusions about the nature of the two different protein phases. Materials and Methods Langmuir Trough and Solutions. The experiments were performed at room temperature in a Langmuir trough, 5-6 mm deep, which is equipped with two movable barriers. The surface pressure and the movement of the barriers are computer controlled. The subphase for all experiments contained a buffer of 10 mM Hepes, 250 mM NaCl, and 10 µM EDTA (all from Sigma) at pH ) 7.8. The buffer was shaken with chloroform to remove organic contaminations, and after separation the aqueous part was shaken with hexane to extract traces of chloroform. The lipid solution used was DPPE-X-Biotin (Molecular Probes) dissolved in chloroform/methanol (3:1) (HPLC grade, Aldrich). Streptavidin, which was purchased from Boehringer Mannheim, is lyophilized from water solution. Some of the lots used were numbers 13381661-33, 13381664-33, and 13381668-33. It was core streptavidin, and mass spectrometry analysis yielded a molecular weight of about 52 000. Streptavidin was dissolved in the buffer described above. The protein concentration was determined by absorption spectroscopy at 280 nm with an absorption coefficient for the protein of 136 000 M-1 cm-1 . After spreading, the lipid was compressed to a surface pressure of 28 mN/m, and the protein solution was injected through the surface into the subphase with a micropipetter. After immediate recompression to 28 mN/m to compensate for the small disturbance due to the injection, the monolayer was kept at a constant surface area. The surface pressure dropped no more than 3 mN/m during extended periods of time. The final protein concentration in the trough ranged from 1.8 to 11 µg/mL which corresponds to 33-220 nM, respectively. Protein Labeling and Fluorescence Microscopy. For the BAM-fluorescence coincidence control experiment, streptavidin was labeled with FITC (Molecular Probes). For this procedure, the protein as well as the FITC was dissolved in a buffer of 50 mM sodium bicarbonate (Baker Chemicals) and 150 mM NaCl at pH ) 9.0 . The buffer was cleaned as described above. Both solutions were mixed in a ratio of protein/FITC ≈ 3/2 (wt/wt) and incubated at room temperature for 30 min. This mixture was passed over a PD-10-desalting column (Pharmacia) to separate the protein from the free FITC. The labeling ratio was determined to be two to three FITC molecules per protein by absorption spectroscopy at 280 and 496 nm with absorption coefficients for FITC of 74 000 M-1 cm-1 (496 nm) and 25 000 M-1 cm-1 (280 nm). A Nikon epi-fluorescence microscope with an extra long working distance objective (40×, 5 mm) was used. It was focused by moving the optics rather than the sample. The image was recorded by an intensifier with an optically coupled CCD camera. Images were processed as described below. Brewster Angle Microscopy. The home built Brewster angle microscope (BAM) is mounted above the Langmuir trough (Figure 1). The incoming He-Ne laser light of 10 mW is

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Figure 2. Time sequence of the growth of unlabeled streptavidin crystals underneath a DPPE-X-biotin monolayer. The protein aggregates identified with the well-known H-shaped two-dimensional streptavidin crystals are bright due to their higher protein density compared to that of the surrounding. The crystals grow in size, and the number of crystals increases over a period of (a) 8, (b) 16, and (c) 59 min after their first appearance. p-polarized by a Glan-Thompson polarizer (intensity ratio 10-5) at an angle of incidence of 53.12° with respect to the surface normal (Brewster angle for the air-water interface). The intensity reflected from the plain air-water interface at the Brewster angle is at the detection limit. If a monolayer covers the surface, the reflected intensity increases and is collected by a lens system to form an image on a CCD camera. The extra long working distance of our BAM (about 50 mm) allows enough space to combine BAM with other techniques for simultaneous observations from the same surface spot. Details of this setup will be published elsewhere.33 The images from the CCD camera are captured with “NIHImage”. We use a SG-9 Scion Capture Card (Scion Corp.) which digitizes the image to 8 bit grayscale and allows on-line background subtraction. In some cases the images were recorded on S-VHS and processed later. For all intensity measurements the background option was disabled. Displayed images in this paper are noise filtered. As the light collected by the BAM is very sensitive to the water level, the water level was automatically kept at a constant height by a home built level control. The absolute intensity of reflected p-polarized light was stable and reproducible from experiment to experiment within a range of about (3 grayscale units out of 256.

Results and Discussion 1. Streptavidin Crystals Visualized by BAM. Streptavidin was chosen as our model protein to explore the extent to which BAM can be utilized for a quantitative study of protein adsorption and aggregation processes at interfaces. A biotinylated monolayer is compressed to a surface pressure of π ) 28 mN/m, and streptavidin is then injected underneath. The BAM images from the interface are featureless prior to and shortly after streptavidin injection. Their grayscale increases over time, indicating surface binding of the protein. Finally, regularly shaped aggregates appear bright on a less intense background as seen in Figure 2. The aggregates grow in size as the system equilibrates. The grayscales of the aggregates and of the surrounding phase are well above the grayscale of the pure lipid monolayer, indicating that protein is bound to both phases. The aggregates move on the less bright background, driven by convection indicating that the surrounding phase is fluid. The aggregates of unlabeled streptavidin visualized by BAM are similar in shape to those typically observed by fluorescence microscopy of photolabeled streptavidin, and it is well-established that the latter are crystalline.10 In a control experiment, we photolabeled streptavidin and brought BAM and fluorescence microscopy in coincidence so as to probe the same surface spot simultaneously by the two techniques. The bright aggregates visualized in BAM were of the same shape and size and also showed polarized fluorescence which proves that the aggregates seen in BAM are indeed identical with two-dimensional streptavidin crystals. On the basis of these observations, we identify the regular shaped, bright aggregates seen by BAM as two-dimensional protein crystals. (33) Schief, W. R., Jr.; Frey, W.; Vogel, V. Manuscript in preparation.

Figure 3. Comparison of the theoretical reflected intensities for different surface layer systems at the air-water interface as a function of the refractive index of the layer, left, as schematically depicted on the right. Curves (a) and (b) compare a lipid monolayer of 17 and 25 Å thickness, respectively, and curve (c) shows a streptavidin layer bound to a lipid layer of 17 Å. For comparison to (c), the equivalent dependence for s-polarized light, suppressed by 10-5, is given in (d) to show that it contributes only as additive background. Using the Maxwell-Garnett approximation, the refractive index can be converted into protein surface coverage. Refractive index and coverage are nearly linearly related in this case, so that the (c)-curves for the refractive index and the surface coverage are on top of each other.

2. Determination of the Protein Surface Density. The advantage of BAM imaging is that the spatially resolved intensity of the reflected p-polarized light yields quantitative information about the protein density distribution at interfaces, information that cannot be deduced easily from fluorescence microscopy. The change of the p-polarized reflectivity, which depends on the composition of the surface layer, can be described by Fresnel’s equations for a layered system of uniform slabs, each characterized by a uniform thickness and a refractive index.34 Figure 3 displays the calculated intensity dependence of the surface layer on the refractive index and thickness for a layered system as depicted schematically. Due to the suppression of the s-polarized light by 10-5 by the polarizer, the reflectivity for s-polarized light does not change measurably with the optical thickness of the interfacial film for all the systems investigated here. Although intensity measurements reveal only one optical parameter, conclusions on the adsorption process and the process of crystal formation are possible with the usage of parameters determined by other methods. The parameters taken from the literature used to calibrate Irefl are listed in Table 1. The protein surface density distribution within individual BAM images can now be analyzed quantitatively. (34) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North Holland: Amsterdam, 1977.

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Figure 4. Grayscale analysis of a BAM image. (a) A typical BAM image of a crystal. Along the white line the grayscale values are extracted. (b) Extracted grayscale distribution displayed as a function of location on the white line. In comparison, the grayscale of the pure lipid monolayer is about 50. To calculate refractive index and relative protein surface coverage, the average values are taken as indicated by the dashed line. (c) Refractive index and surface coverage for the averaged grayscale distribution as calculated from the Fresnel equations and the Maxwell-Garnett model (eq 4). Table 1. Parameters Used To Calibrate Irefl intensity level free water surface water surface covered with a compressed lipid monolayer (no spacer) water surface covered with a compressed lipid monolayer and a crystalline protein layer

idealized slab system air water air lipid water air lipid protein crystal water

Figure 4b displays an intensity profile across a BAM image (Figure 4a) taken along the indicated white line. Two distinct intensities are found within and surrounding the streptavidin crystals. Both absolute intensities are much higher than that for the lipid monolayer alone. The dotted line in Figure 4b indicates the average along the white line in Figure 4a, within the crystals and the surrounding area. For the quantitative grayscale analysis given below, areas instead of lines are averaged from several regions within an image. The typical noise level is then reduced to about (3 grayscale units. The observed intensities can be converted into a refractive index for the protein layer according to Fresnel’s equations. The absolute intensity measured by the camera is given by

Irefl ) (I0RF)TCCD - g0

(1)

Here Irefl is the reflected intensity in units of grayscale (0-255), TCCD is the digitization function, which transfers the part RF of the incoming intensity I0 which is reflected from the interface into grayscale units, and g0 is the black level. The reflected intensity of the pure air-water interface is not used as black level for calibration in order to exclude errors due to the limited sensitivity of the CCD camera. The Fresnel coefficient RF is assumed to be RFp only and any RFs, which is suppressed by 10-5, is neglected.

d)∞ d)∞ d)∞ d ) 17 Å d)∞ d)∞ d ) 17 Å d ) 45 Å d)∞

ref n ) 1.0 n ) 1.333 n ) 1.0 n ) 1.50 n ) 1.333 n ) 1.0 n ) 1.50 n ) 1.45 n ) 1.333

14 14, 31

We found that the measured intensities for one system are very stable over time and reproducible between different measurements. If the curves for different experiments have slightly different intensities at the time of injection, this offset is subtracted when intensities are compared. The stability of the absolute grayscale between experiments allows, in principle, calibration of the intensity for other systems for which not so much information is available. Besides assuming the parameters in Table 1, the only additional assumption is that the thickness of the protein slab is constant. This is reasonable, since the protein streptavidin is very compact and does not change its overall shape significantly upon binding.7 The left axis of Figure 4c shows the refractive index calculated from the averaged intensity distribution displayed in Figure 4b. To interpret the calculated refractive indices for the protein layer, the relationship between refractive index and protein surface density is modeled by the MaxwellGarnett theory.34,35 The Maxwell-Garnett model assumes the individual surface-bound proteins to be spherical dipole scatterers separated by bulk water and calculates an effective refractive index neff based on a mean field model. It also assumes a quasistatic limit of very large wavelength compared to the protein diameter. The Maxwell-Garnett (35) Maxwell Garnett, J. C. Philos. Trans. R. Soc. London 1904, 203, 385; 1906, 205, 237.

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For the determination of the protein volume fraction Θ′, the refractive index for the pure protein layer nprot is not known. Since protein crystals contain a high amount of water (≈50 vol%),14 which mostly has bulk properties, extrapolating nprot from the crystals using eq 2 strongly depends on the amount of water with bulk properties inside the crystal. We therefore relate our results for the surface density of the noncrystalline protein σnoncryst to the surface density of the protein in the crystal σcryst:

Θ)

σnoncryst Θ′noncryst ) σcryst Θ′cryst

(3)

with Θ as the relative protein surface density. Reformulation of eq 2 yields:

neff2 - nw2

nprot2 - nw2 ) Θ′ neff2 + 2nw2 nprot2 + 2nw2 Figure 5. The Maxwell-Garnett model (eq 2) is used to calculate the effective refractive index of a protein-water slab, neff. As examples we have calculated the dependence of neff on the volume fraction of protein in the protein-water slab for four assumed refractive indices nprot(Θ′ ) 1) of 1.64, 1.57, 1.53, and 1.45 (solid lines). The two curves for protein embedded in water and water embedded in protein are displayed and coincide for a given nprot. Each value of nprot corresponds to a certain fraction of protein inside the crystal which is given by the value of Θ′ at the known effective refractive index for a streptavidin crystal neff ) ncryst ) 1.45 (dashed lines).

theory specifies the effective refractive index for proteins embedded in water as

neff2 - nw2

nprot2 - nw2 ) Θ′ neff2 + 2nw2 nprot2 + 2nw2

(2)

Here nprot is the unknown refractive index of a hypothetical protein monolayer which contains no water; nw is the refractive index of water which occupies (1 - Θ′) of the surface, and Θ′ is the volume fraction of protein in the protein-water slab. The Maxwell-Garnett model does not hold generally if the density of the embedded particles becomes too high, if the particles are arranged in an array, or if the particles are nonspherical.36 Another restriction of the theory is that the effective refractive index for medium 2 embedded in medium 1 (2-in-1) is not the same, in general, as that for medium 1 embedded in medium 2 (1-in-2). However, in the case that both the embedding medium and the embedded medium are isotropic and nonabsorbing (the dielectric constant , scalar and real), it has been shown from general principles37,38 that the effective refractive indices as a function of composition Θ′ for systems 1-in-2 and 2-in-1 form the upper and lower limits of all physically achievable solutions, independent of the topology of the system.39 In our situation of proteins embedded in water, the solutions for 1-in-2 and 2-in-1 fall essentially onto the same curve, as seen from Figure 5. We therefore can determine neff or Θ′ from the Maxwell-Garnett model even though the protein is partly in a crystalline order and the protein molecule is not really a sphere. The model is also valid in this case for high protein concentrations in the protein-water slab. (36) Fu, L.; Macedo, P. B.; Resca, L. Phys. Rev. B 1993, 47 (20), 13818. (37) Hashin, Z.; Shtrikman, S. J. Appl. Phys. 1962, 33 (10), 3125. (38) Brown, W. F. J. J. Chem. Phys. 1955, 23 (8), 1514. (39) Lamb, W.; Wood, D. M.; Ashcroft, N. W. Phys. Rev. B 1980, 21 (6), 2248.

)

nprot2 - nw2 Θ′ Θ′cryst Θ′cryst n 2 + 2n 2 prot

2

neffcryst - nw

2

w

ncryst2 - nw2

)Θ ≡Θ neffcryst2 + 2nw2 ncryst2 + 2nw2

(4)

This is the Maxwell-Garnett relation which allows us to calculate the relative protein surface density Θ from neff as deduced from the BAM grayscales and from the known value of ncryst. The relative protein surface density Θ calculated from the intensity distribution of Figure 4b using eq 4 is displayed in Figure 4c on the right axis. As can be seen also from Figure 3 the relation between refractive index and surface coverage is practically linear for the system investigated here. 3. Time Dependence of Streptavidin Crystallization. Following the above analysis, we can quantitatively probe the protein adsorption in a spatially resolved fashion. Figure 6 shows reflected intensity versus time extracted from BAM images taken from the free water surface (arrow a) and the biotinylated monolayer after spreading (arrow b) and after compression (arrow c) for times t < 0, as well as the protein adsorption after injection of streptavidin for times t > 0. The presence of the lipid monolayer changes the reflectivity of the air-water interface only slightly in comparison to the large increase due to the adsorption of streptavidin to the interface. A typical sigmoid intensity curve (dark line) characterizes the adsorption process. The time and intensity at which crystals are first seen is indicated by the dashed line. After the occurrence of the first crystals, the reflected intensities of the crystalline (gray line) and the noncrystalline areas (dark line) are analyzed separately. The difference in intensity between the noncrystalline protein layer and the crystalline domains is large compared to that of the lipid monolayer alone. Adsorption curves were fitted to y ) a + b(1 exp(-(cx)n)) as indicated by the line, in order to focus the eye on the general behavior of the adsorption curves. For comparison, Figure 6 shows also the original scatter of the data. The fluctuations in the reflected intensity are mainly due to lateral density nonhomogenities in the protein layer. Figure 7a compares the change of the reflected intensity over time for different protein concentrations in the bulk. Again the dark and the gray bold lines represent reflection from the noncrystalline and the crystalline phases, respectively. The sigmoid shape of the adsorption curve

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Figure 6. Reflected intensity versus time for the adsorption of streptavidin to a DPPE-X-biotin monolayer as deduced from a BAM image sequence. The arrows indicate the signal from (a) the free air-water interface, (b) the air-lipid-water interface at π ≈ 20 mN/m, and (c) after compression of the lipid to π ) 28 mN/m. The time t ) 0 indicates the injection of streptavidin to a final concentration of 33 nM. The reflected intensities for the noncrystalline phase (+) are connected by the dark fitted line and the gray line for the crystalline phase (o). The dotted lines mark the first occurrence of the crystals seen with BAM.

for the noncrystalline phase is preserved for all bulk concentrations. A quantitative analysis of the adsorption kinetics, however, is not intended since the transport mechanism of the proteins to the interface is not well defined in our experiments. Initially, the reflected intensity remains constant after protein injection. Then the intensity rises, indicating protein binding to the interface. The delayed onset of the intensity rise as well as the slope of the adsorption curve depends on the protein bulk concentration. The first crystals appear before the intensity of the noncrystalline phase levels off. At the first appearance of the crystals, the intensity of the crystalline phase is higher than that of the noncrystalline surrounding. It is noteworthy that the intensity of the noncrystalline phase at this point is the same for all bulk concentrations, the value being 84-89 grayscale units (dashed lines). This grayscale translates into a relative surface density of 75% of the crystal density. A critical

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surface density is hence required to induce the growth of two-dimensional streptavidin crystals. As time progresses, the curves for the noncrystalline phase level off to final values below (curve a) or above (curves b and c) those of the crystalline phase for low- or high-protein bulk concentration, respectively. This is astounding since it implies that streptavidin can be packed more densely in the noncrystalline than in the crystalline phase. A maximum of about 110% of the crystal density is reached for bulk concentrations above 40 nM. Under such conditions, the crystals appear dark on a bright background since they are surrounded by more dense material as seen in Figure 8. The BAM contrast is inverted. The intensity of the crystalline regions, however, is constant over time as well as between different measurements within experimental error. This indicates that the protein density inside the crystals is unchanged over time and does not depend on either the protein bulk concentration or the density in the surrounding phase. In contrast, the final intensity of the noncrystalline protein phase strongly depends on the protein bulk concentration. Following the analysis given above, we can determine the refractive index and the corresponding relative surface density over time as displayed in Figure 7b. In the cases of contrast inversion, the relative protein density in the noncrystalline phase is larger than 1, suggesting a lower water content in these areas. A change in refractive index for the pure protein, which would mean a compression of the individual protein molecule, can be excluded. A second protein layer can be excluded as well, since the total intensity increase is much too small for a continuous second layer. A discontinuous second layer would grow with the addition of protein, which contradicts our finding of an upper limit on the intensity and a constant intensity of the crystals. Additionally, streptavidin shows a very low unspecific adsorption. To better understand the behavior of the noncrystalline phase, the surface binding of streptavidin was studied under conditions that are known for not producing streptavidin crystals. No crystallization has been observed on an otherwise identical buffer if it does not contain NaCl,10 a finding that is confirmed by our BAM studies. Streptavidin, however, binds to the interface on such a salt-free solution as indicated by the spatially homogeneous increase of the reflected intensity. Figure 9 shows the adsorption isotherms for three streptavidin bulk concentrations with and without the presence of salt. Intensity and hence protein surface density are lower on salt-free solutions than on standard buffer solutions for

Figure 7. Calculated change in protein refractive index and surface coverage over time for different protein bulk concentrations: (a) 33, (b) 65, and (c) 220 nM. All three experiments show adsorption and crystallization following protein injection. The first appearance of crystals is indicated by the dashed lines. For all concentrations, the crystals first appear once the protein surface density has reached a critical value of approximately 75% of the crystal density. At a bulk concentration of 33 nM, the final coverage of the noncrystalline phase remains below that of the crystalline phase, indicating depletion of protein in the subphase. For the bulk concentrations of 65 and 220 nM, the final noncrystalline coverage exceeds the crystal coverage by no more than approximately 10%, indicating maximum surface coverage.

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Figure 8. Two BAM images corresponding to the data shown in Figure 7b. (a) When the crystals first appear, they are bright surrounded by the darker noncrystalline areas. (b) After the intensity of the noncrystalline areas exceeds the crystal intensity, the crystals appear dark on a bright background; the contrast is inverted. The protein surface density in the noncrystalline phase is higher than that in the crystal.

Figure 10. Pressure-area isotherms for the pure lipid and two lipid-protein systems. In (a) the lipid-protein film is compressed just below the phase transition of the pure lipid, and in (b) it is compressed into the phase transition. The pressure is dominated by the compression of the protein layer as indicated by the lower compressibility (extrapolated dashed lines) compared to that of the pure lipid system. While film (a) is reversible on expansion, film (b) shows the collapse of the protein layer, and the hysteresis is large on expansion. Figure 9. Comparison of the reflected intensity over time on a buffered subphase with and without 250 mM NaCl for different protein bulk concentrations: (A) 33, (B) 65, and (C) 220 nM. Curves a-c and R-γ refer to the salt-containing and salt-free subphases, respectively. The intensities of the crystals are again indicated as gray lines. The protein does not crystallize on low electrolyte concentrations. The intensities in curves β and γ show a slow increase even at advanced equilibration times and approach the saturation intensity of the noncrystalline phase given in γ.

comparable protein bulk concentrations. In the case of high bulk protein concentration, the intensity slowly increases and eventually exceeds the crystal intensity (cf. Figure 9c). Finally, the intensity levels off at about the maximum saturation intensity of the noncrystalline phase of those salt systems which show contrast inversion. Another control with pure DMPE lipid on a standard subphase with 33 nM streptavidin showed an increase of only 5 grayscale units over 3 h (not shown). This agrees with the known fact that streptavidin shows little nonspecific adsorption. 4. Compression of the Protein Layer. Compression and expansion of the protein film provides further insight into the structural properties of the two phases. Figure 10 shows the pressure-area isotherms for the pure biotinylated lipid and two lipid-streptavidin systems. The pure lipid shows a phase transition at a surface pressure of about 32-34 mN/m on the standard buffer. On protein injection underneath the lipid monolayer at π ≈ 28 mN/ m, the surface pressure did not rise. After equilibration for at least 3 h, the monolayer was slowly compressed. The rise of the surface pressure is mainly due to the compression of the protein layer indicated by the much lower compressibility (steeper increase in the π-A isotherm as indicated by the extrapolated dashed line in Figure 10) compared to that of the pure lipid. The compression is reversible if the lipid-protein layer is only compressed to pressures below the lipid phase transition (π e 32 mN/m). If the film is compressed into the lipid phase transition, however, the pressure drops to that of the plain lipid and the expansion shows a large hysteresis. In the case of reversible compression to pressures below

Figure 11. The reflected intensities of the noncrystalline and crystalline phases on monolayer compression and expansion. The reflected intensity of the noncrystalline phase increases when the monolayer is compressed from 28-32 mN/m, while it is constant for the crystalline phase. The process is fully reversible on expansion, since the pressure never exceeded the onset of the lipid phase transition. The inset shows streptavidin crystals (now dark) surrounded by more densely packed noncrystalline protein in the compressed state.

the lipid phase transition, Figure 11 shows contrast inversion. The reflected intensity and with it the surface density of the noncrystalline phase exceeds that of the crystalline phase during compression. The film was compressed from 106 to about 94 Å2 per lipid molecule. The insert of Figure 11 shows a BAM image of the compressed state at π ≈ 31 mN/m. While the crystalline protein areas do not change in appearance and intensity, the noncrystalline phase is now brighter. The surface pressure increase (Figure 10) as well as the contrast inversion is fully reversible on expansion, leading to the same surface density levels as prior to compression (Figure 11). A sequence of BAM images following the same crystalline domain on expansion is given in Figure 12.

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Figure 12. Series of images recorded from the same surface spot on monolayer expansion from 31 to 28 mN/m. The contrast gradually converts back to bright crystals surrounded by a less densely packed noncrystalline phase.

The crystals appear darker on a bright background at the pressure of 32 mN/m (image 1), and then the contrast fades away (images 2-5) and finally establishes the original bright crystals on a darker background as the pressure reaches 28 mN/m (image 6). While the intensity of the bright areas decreases during expansion, the intensity of the crystals remains constant. In the case of irreversible compression to pressures above the lipid phase transition, the crystalline and noncrystalline protein phases both collapse, and the pressure drops to that of the lipid. On expansion, the protein crystals disappear, but bright lines of highly compressed, noncrystalline protein remain to be seen. Discussion and Conclusions Our results show that BAM is well suited to probe protein adsorption and aggregation processes at interfaces in situ. The protein surface density can be determined quantitatively without the use of a fluorescent dye and associated quenching effects. Streptavidin was chosen as a model protein since its two- and three-dimensional crystals are well characterized in the literature. BAM combined with a Langmuir trough provides new insight into the dynamics of the crystallization process as well as into the mechanical properties of the lipid-protein film. Gaining further insight into the mechanical properties of surface-bound proteins is of special interest since protein crystals are known to possess a relatively high water content. Streptavidin crystallized in three dimensions includes a water content of about 50 vol% per protein in the unit cell.9 Two-dimensional streptavidin crystals transferred to solid supports are arranged into a rectangular unit cell which includes a large fraction of “voids”, as demonstrated by electron diffraction.10 X-ray diffraction at the air-water interface determined the amount of water in the protein layer to be 1600-2000 water molecules per protein (48-60 vol%).14 It was suggested that the two-dimensional protein crystals are compressible, thereby continuously excluding water molecules.17,18 Our results show that the water exclusion is very different for the two protein phases. The noncrystalline phase can be mechanically compressed to surface concentrations that lie above that of the crystalline phase. The compression of the noncrystalline phase is fully reversible if the pressure of the onset of the lipid phase transition is not exceeded. The behavior of the crystals upon compression is clearly distinct from that of the noncrystalline phase. The grayscale, and corresponding refractive index, of the crystalline phase remains constant even after the protein density of the surrounding exceeds the crystal density. This implies that the compressibility of the crystalline phase is too low to be detected in our experiments. However, we have not yet been successful

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in capturing sequences of single crystals upon compression over a large compression range in order to verify that the crystal shapes are unaffected by compression. Compression of the surface-bound protein layer above the phase transition of the DPPE-X-biotin monolayer, namely above π ) 32 mN/m, leads to an irreversible destruction of the streptavidin crystals which is then accompanied by changes in the crystal shape and intensity. Surface densities that exceed that of the crystalline phase can be obtained not only by compression but also by adsorption from the bulk for sufficiently high streptavidin bulk concentrations. The saturation limit of the noncrystalline phase on adsorption is the same on either sodium-containing or sodium-free buffer solutions even though it is known for the latter case that streptavidin does not crystallize. The refractive index of the fully saturated noncrystalline phase is about 10% larger than that of the crystalline phase. The protein molecules are apparently packed more densely within the fully saturated noncrystalline phase than within the crystals. This implies that the water content of the noncrystalline phase is gradually reduced to values below the water content of the crystals as the noncrystalline protein density approaches saturation. The enhanced refractive index of the fully saturated noncrystalline phase translates into an increased protein surface density of about 10% with respect to the crystalline phase. These results could explain findings reported with fluorescence-labeled streptavidin, in which a rim with enhanced fluorescence intensity surrounding the crystals was observed.13,40 Upon adsorption of streptavidin to compressed biotinylated lipid monolayers, the first crystals observed in BAM appear at a surface density of about 75% relative to that of the crystalline phase. Our quantitative BAM results of the streptavidin crystallization process call for a discussion of the nature of the noncrystalline-tocrystalline phase transition within the surface-bound protein layer. The finding of a critical surface density at which crystals first appear in BAM may be taken as an indication of a first-order phase transition. The protein surface density is the order parameter. It increases continuously on adsorption until it is discontinuous at the critical surface density. The critical surface density is therefore expected to be independent of the protein bulk concentration, as observed experimentally. The formation and dissolution of streptavidin crystals on compression and expansion of the lipid-protein layer also support the notion of a first-order phase transition.41 Contradicting this interpretation, however, is the fact that the protein layer does not establish a true first-order coexistence of the noncrystalline and crystalline phases when the streptavidin surface density rises above the critical value of about 75%. Instead, the density of the noncrystalline phase keeps increasing continuously, and the crystals stop growing before the adsorption process reaches saturation. The surface density of the crystalline phase remains constant over time, and the density of the noncrystalline phase can finally exceed that of the crystalline phase. All the BAM data gathered above the critical surface density of about 75% can consistently be explained if we assume that our streptavidin solution (Boehringer Mannheim) contains more than one type of high-affinity streptavidin, some fraction of which is biotin bound but not capable of two-dimensional crystallization. This protein fraction would be enriched in the noncrystalline phase due to exclusion from the crystals. The critical (40) Hoffmann, M.; Mu¨ller, W.; Ringsdorf, H.; Rourke, A. M.; Rump, E.; Suci, P. A. Thin Solid Films 1992, 210/211, 780. (41) Schwinn, T.; Heyn, S. P.; Egger, M.; Gaub, H. E. In Thin Films; Ulman, A., Ed.; Academic Press: San Diego, 1995.

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surface density required for further crystal growth and nucleation would thereby not be constant but rise as this protein fraction gets enriched in the noncrystalline phase. A detailed analysis of this effect is in progress.42 In conclusion, our data suggest that BAM has all the credentials to develop into a most powerful tool for the controlled growth of two-dimensional protein crystals at interfaces. BAM could make a significant contribution to structural biology in the difficult search for the right conditions which allow the two-dimensional crystallization of membrane-anchored or ligand-bound proteins. BAM can visualize in situ whether or not regularly shaped aggregates are being formed at the interface and give information about their size and number. Since photolabeling is not required, potential difficulties with a label attachment and potential steric hindrances are avoided. (42) Schief, W. R., Jr.; Frey, W.; Chilkoti, A.; Stayton, P. S.; Vogel, V. Manuscript in preparation.

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Another application of BAM may be in biotechnology and material sciences where the goal is to maximize the size of protein crystals such that they can be used as twodimensional templates. The ability to measure the critical protein surface density which is required to induce crystal nucleation is most desirable since it allows the optimization of growth conditions. Acknowledgment. We thank A. Chilkoti and P. Stayton for the many insightful discussions on streptavidin and the streptavidin crystallization processes. We gratefully acknowledge the following support: W.F. by a Feodor Lynen Fellowship (Alexander von Humboldt Foundation), W.R.S. by a NIH Training Grant (GM 08437), and V.V. by the NIH First Award (1R29 GM 49063-01A1). Additional funding was provided by PNL-Battelle (234509), The Whitaker Foundation, and NASA (NAG8-1149). LA9505374