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Molecular Arrangement in Two-Dimensional Streptavidin Crystals Szu-Wen Wang, Channing R. Robertson, and Alice P. Gast* Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025 Received August 14, 1998. In Final Form: December 4, 1998 We studied the molecular arrangement of two-dimensional streptavidin crystals at the air-water interface over a range of pH values. We quantified the varying amounts of coexisting P1, P2, and C222 crystals in the different morphologies observed at pH 4.5-6.5. Chiral, needlelike crystals at pH 4.5 consist of P1 crystals with frequent line defects. Larger chiral domains near pH 5 are essentially all P1 coexisting with a small amount of P2, whereas at slightly higher pH values (near pH 5.5), H-shaped domains contain 4 times as much P1 coexisting with a P2/C222 mixture. Morphologies intermediate to these shapes exhibit intermediate compositions. Between pH ∼6-7, crystals all display a characteristic dendritic-X morphology, but arrangement at the molecular level is quite different compared with lower pH values. Crystals are mostly P2 in symmetry near pH 6, but at pH 7 and above, crystals have C222 symmetry. Coexistence of P2 and C222 crystals occurs at intermediate pH values. We determined the orientation and arrangement of streptavidin molecules in P1, P2, and C222 crystals relative to the directions exhibiting faster growth. The direction of faster growth in P1 crystals includes both interactions between biotin-free subunits and interactions between biotin-bound subunits. In the P2 arrangement, growth in the direction of intermolecular biotin-free subunits is preferred, whereas growth is faster along the biotin-bound direction of C222 crystals. We developed a model of the molecular arrangement for the observed solid-phase coexistence in these crystals.
Introduction The protein streptavidin has been widely studied1-6 and exhibits many unusual properties. It shows high binding affinity to the vitamin biotin (10-15 M),7 displays a tetrameric geometry such that its two pairs of biotin binding sites exist on opposite faces of the protein,1 and is stable over substantial variations in pH2,8 and temperature.9 These attributes make streptavidin and its homologous protein avidin useful in many applications,6 from purification processes10 to immunoassays.11 Another application of the streptavidin-biotin system encompasses the study of self-assembling protein arrays.12-18 Streptavidin molecules bound to a monolayer * Corresponding author. (1) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85. (2) Green, N. M. Biochem. J. 1966, 101, 774. (3) Green, N. M. Adv. Protein Chem. 1975, 29, 85. (4) Green, N. M. Methods Enzymol. 1990, 184, 51. (5) Chaiet, L.; Wolf, F. J. Arch. Biochem. Biophys. 1964, 106, 1. (6) Bayer, E. A.; Wilcheck, M., Eds. Avidin-Biotin Technology; Academic Press: San Diego, 1990; Vol. 184. (7) Bayer, E. A.; Ben-Hur, H.; Wilchek, M. Methods Enzymol. 1990, 184, 80. (8) Hofmann, K.; Wood, S. W.; Brinton, C. C.; Montibeller, J. A.; Finn, F. M. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 4666. (9) Bayer, E. A.; Ben-Hur, H.; Wilcheck, M. Methods Enzymol. 1990, 184, 80. (10) Finn, F. M.; Hofmann, K. Methods Enzymol. 1990, 184, 244. (11) Ternyck, T.; Avrameas, S. Methods Enzymol. 1990, 184, 469. (12) Ku, A. C.; Darst, S. A.; Kornberg, R. D.; Robertson, C. R.; Gast, A. P. Langmuir 1992, 8, 2357. (13) Ku, A. C.; Darst, S. A.; Kornberg, R. D.; Robertson, C. R.; Gast, A. P. J. Phys. Chem. 1993, 97, 3013. (14) Wang, S.-W.; Poglitsch, C. L.; Yatcilla, M. T.; Robertson, C. R.; Gast, A. P. Langmuir 1997, 13, 5794. (15) Frey, W.; Schief, W. R.; Vogel, V. Langmuir 1996, 12, 1312. (16) Frey, W.; Schief, W. R.; Pack, D. W.; Chen, C.-T.; Chilkoti, A.; Stayton, P.; Vogel, V.; Arnold, F. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4937. (17) Blankenburg, R.; Meller, P.; Ringsdorf, H.; Salesse, C. Biochemistry 1989, 28, 8214. (18) Vaknin, D.; Kjaer, K.; Ringsdorf, H.; Blankenburg, R.; Piepenstock, M.; Diederich, A.; Lo¨sche, M. Langmuir 1993, 9, 1171.
of biotinylated lipid at an air-water interface can form two-dimensional (2D) protein crystals.12,17,19 We use this system to examine interactions among proteins to obtain a better understanding of the effects they have on 2D self-assembly phenomena. This information is needed to grow larger, more ordered crystals for protein structure analysis.20,21 Additionally, this work has implications in the development of interfacial technologies such as biosensors and biomaterials,22-24 which require presentation of a protein binding site or functional group in a highly ordered and oriented configuration. The growth of streptavidin crystal morphologies in a Langmuir trough can be monitored by different optical techniques,15,17,19 and the molecular arrangement of these crystals can be analyzed with transmission electron microscopy (TEM) and electron diffraction.13,14,19,25 Twodimensional ordered arrays of streptavidin molecules selfassemble in three distinct crystalline arrangements: P1 at pH 4,25 P2 at pH 5.5,14 and C222 at pH 7.19 Various crystal morphologies occur at different pH ranges, and these differences in morphologies reflect the molecular packing.14,26 Needlelike morphologies at pH 4 consist of crystals with P1 symmetry, and dendritic-X domains at pH > 7 contain crystals with C222 arrangement.14,26 The (19) 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, 387. (20) Leuther, K. K.; Bushnell, D. A.; Kornberg, R. D. Cell 1996, 85, 773. (21) Asturias, F. J.; Meredith, G. D.; Poglitsch, C. L.; Kornberg, R. D. J. Mol. Biol. 1997, 272, 536. (22) Koppenol, S.; Stayton, P. S. J. Pharm. Sci. 1997, 86, 1204. (23) McLean, M. A.; Stayton, P. S.; Sligar, S. G. Anal. Chem. 1993, 65, 2676. (24) Mosbach, K., Ed. Immobilized Enzymes; Academic Press: New York, 1976; Vol. 54. (25) Hemming, S. A.; Bochkarev, A.; Darst, S. A.; Kornberg, R. D.; Ala, P.; Yang, D. S. C.; Edwards, A. M. J. Mol. Biol. 1995, 246, 308. (26) Yatcilla, M. T.; Robertson, C. R.; Gast, A. P. Langmuir 1998, 14, 497.
10.1021/la981038g CCC: $18.00 © 1999 American Chemical Society Published on Web 01/30/1999
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different crystal morphologies between pH 5 and 6 were found to consist of a coexistence of P1 and P2 crystals.14 In this work, we extend the characterization of the solidphase coexistence and quantify the coexistence ratios of the various intermediate crystal domains. Additionally, we analyze the three types of crystals (P1, P2, and C222) and coexisting crystals in order to correlate the orientation of the molecules and biotin binding with the crystal growth directions. The molecular features of crystal coexistence and protein arrangement are especially important for the study of the intermolecular interactions promoting crystallization. Manipulation of these interactions27-29 requires knowledge of important contact regions.30 These can be inferred from molecular details and crystal growth directions. Methods and Materials 2D Crystallization on a Langmuir Trough. We spread a monolayer using a 1:10 lipid mixture containing N-(6-((biotinoyl)amino)-hexanoyl)dipalmitoyl-L-R-phosphatidyl-ethanolamine (Molecular Probes, Eugene, OR) and dioleoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) to a surface pressure of 25-30 mN/m on a miniature Langmuir trough. A 1:2 mixture of fluorescently labeled streptavidin (Boehringer Mannheim, Indianapolis, IN) and unlabeled noncrystallizable avidin (Sigma, St. Louis, MO) was injected, and crystallization was monitored in situ with epifluorescence microscopy. Details are given in Ku et al.12 and Wang et al.14 Crystal Transfer onto TEM Finder Grids. TEM finder grids (Ernest F. Fullam, Inc., Latham, NY) were coated with a thin layer of carbon.31 To preserve crystal morphology during and after transfer onto these grids, glutaraldehyde (Sigma, St. Louis, MO) was gently injected into the buffer subphase to a final concentration of 0.5% and allowed to diffuse through the buffer and react with protein for at least 4 h. Carbon-coated TEM grids were placed onto the surface of the trough. We observed that grids removed from the Langmuir trough surface at this point often contain fragmented crystals. In addition, a second layer of protein can fold over and adhere to the grid, and this layer is inverted relative to the first layer, as would be expected in Langmuir-Blodgett deposition. In chiral domains, this effect gives crystals a handedness opposite to that found for a singlelayer deposition. To ensure that only a monolayer of crystal was deposited onto the grid and that the chirality and morphology of the domains were preserved, a piece of backing paper from Parafilm (American National Can, Greenwich, CT) was placed over the entire surface of the trough and raised when the monolayer and TEM grids adhered to the paper. Protein in the monolayer surrounding the carbon-coated grid therefore deposited onto the paper instead of forming a second protein layer on the grid. Grids were washed with 5 µL water and negatively stained with 5 µL 1% uranyl acetate for 1 min as described in Ku et al.13 The location of crystals on the grid was documented in fluorescence micrographs. Crystals were visualized by epifluorescence microscopy either through the carbon-coated grid before transfer out of the trough or after the grid was washed with water and dried. In the latter case, rehydration of the crystals on the grids was necessary before negative staining to obtain good contrast under the TEM. Grids were rehydrated by placing a 5-8 µL drop of buffer on the surface of the grid and allowing this to remain in a hydrated container for 2 h. Another 5 µL water wash was applied to the grid before negative staining. (27) Sano, T.; Pandori, M. W.; Chen, X.; Smith, C. L.; Cantor, C. R. J. Biol. Chem. 1995, 47, 28204. (28) Chilkoti, A.; Schwartz, B. L.; Smith, R. D.; Long, C. J.; Stayton, P. S. Bio/Technology 1995, 13, 1198. (29) Edwards, T. C.; Koppenol, S.; Frey, W.; Schief, W. R.; Vogel, V.; Stenkamp, R. E.; Stayton, P. S. Langmuir 1998, 14, 4683. (30) Salemme, F. R.; Genieser, L.; Finzel, B. C.; Hilmer, R. M.; Wendoloski, J. J. J. Cryst. Growth 1988, 90, 273. (31) Bozzola, J. J.; Russell, L. D. Electron Microscopy; Jones and Bartlett Publishers: Boston, MA, 1992.
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Figure 1. Technique for determining molecular arrangement relative to crystal morphology. Crystalline domains are photographically documented on or through the carbon-coated TEM finder grid using fluorescence microscopy. These regions are observed with TEM, and regions near grid square edges are recorded on electron micrographs. Crystalline domains are then analyzed by electron diffraction. Correlation of Morphology with Molecular Arrangement. Electron micrographs of the crystals were obtained with a Philips EM400 at a magnification of 36 000×. Regions for study were identified from photographs of fluorescent crystals on the carbon-coated finder grids, and these same regions were then observed with TEM (Figure 1). This technique allowed us to observe the molecular packing arrangement in different regions of the various macroscopic morphologies. In addition, by noting the direction in which the micrograph was taken (by including a gridsquare edge in the micrograph), the molecular orientation of the protein was determined relative to the crystal domain morphology. This enabled us to conclude the directionality of crystal growth relative to the positions of the bound and unbound biotin-binding sites in streptavidin. Electron diffraction analysis was performed as described by Amos et al.,32 Stewart,33 and in our previous work14 and was performed to reliably discern and identify the three crystal forms. As an orientational reference, a computer-generated array of a chiral object was analyzed with the image digitization and processing protocol, and the resulting orientation was recorded at each step to document the orientation changes. Calculation of Coexistence Ratios. We quantified coexistence ratios of certain distinct morphologies. Micrographs of coexisting crystalline areas were scanned (Leaf Systems, Inc., Southborough, MA), digitized (Adobe Systems, Inc., San Jose, CA), and imported into NIH Image software (version 1.61). Crystals with P2 and C222 symmetry were visually distinguished from P1 crystals, and these areas were measured using the “measurement” macro in NIH Image. Although P1 crystals appear quite visually distinct from P2 and C222 crystals, P2 and C222 forms appear similar to each other. Several (∼15) different regions 1.1 µm2 in extent within individual morphological crystal domains were processed and averaged together to obtain the coexisting ratio values.
Results and Discussion Morphologies and Coexistence Ratios at Various pH Values. We chose distinct morphologies (needles, chiral needles, inverse S’s, H-shaped crystals, and dendritic X’s) for quantifying the coexisting ratios. We found that different morphologies seen between pH 4 and 7.5 consist of different ratios of P1, P2, and C222 crystals (Figure 2). Experiments without avidin gave similar morphologies and ratios, indicating that avidin does not interfere with morphological development or coexistence values. In addition, crystal morphologies were invariant in trials using nonlabeled streptavidin and labeled avidin, (32) Amos, L. A.; Henderson, R.; Unwin, P. N. T. Prog. Biophys. Mol. Biol. 1982, 39, 183. (33) Stewart, M. J. Electron Microsc. Tech. 1988, 9, 301.
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Figure 2. Crystal morphologies at various pH values and their corresponding coexistence ratios. (A) Needlelike domains at pH 4 contain P1 crystals. (B) Chiral needlelike domains at pH 4.5 consist of P1 crystals with frequent line defects. (C) Chiral inverse-S domains at pH ∼4.8-5.5 contain 93 ( 4% P1 crystals coexisting with 7 ( 4% P2 crystals. (D) H-shaped domains at pH ∼5.3-5.7 consist of 21 ( 10% P2 and C222 crystals and 79 ( 10% P1 crystals. (E) Dendritic-X domains at pH ∼5.7-7.5 consist of various crystal ratios ranging from ∼100% P2 to ∼100% C222.
showing that the fluorescent tag does not appear to affect our results. Intermediate Morphologies Have Different P1/P2 Ratios at pH 4.5-5.7. As previously described,14,25,26,34 lowering the pH of the subphase protonates aspartates and glutamates at crystal-contact regions and causes a transformation to the more densely packed P1 crystal. At pH 4, the needlelike crystals consist almost entirely of molecules ordered in a P1 arrangement (Figure 2A).14,26 Crystals at pH 4.5 also contain proteins in a P1 lattice, but frequent line defects break the regularity of this lattice, and the resulting morphology corresponds to chiral needles with slightly curved ends (Figure 2B). As we increase the pH to ∼4.8-5.5, the line defects give way to a coexistence of 7 ( 4% P2 and 93 ( 4% P1 crystals, yielding large chiral inverse-S-shaped domains as previously described14 (Figure 2C). H-shaped domains contain an average of 21 ( 10% P2 and C222 and 79 ( 10% P1 (Figure 2D, Figure 3); coexistence of all three crystal forms is seen in the morphology, which exists near pH ∼5.3-5.7. Morphologies intermediate to those we chose to quantify appear to have intermediate ratios, as expected. For example, coexistence results for inverse-S domains with split ends, which appear to be an intermediate form between inverse-S and H morphologies, gave values of 10 ( 8% P2/C222 and 90 ( 8% P1. Dendritic-X Morphologies Have Different C222/ P2 Ratios at pH 5.7-7.5. Dendritic-X crystals exist between pH ∼5.7 and ∼7.5 (Figure 2E). In this regime, one cannot infer the crystal lattice from the morphology alone. Although the morphology appears to be similar (34) Yatcilla, M. T. Influence of pH on Two-Dimensional Crystals of Streptavidin; Stanford University: Stanford, 1998.
throughout this pH range, molecular differences are evident. Near pH 5.7, crystal domains appear to consist entirely of molecules in P2 arrangement, whereas all crystals above pH 7 are entirely C222. Coexistence is also observed in this range, with a higher ratio of C222/P2 at pH 6.75 than that at pH 6 (Figure 4). Often, P2 regions produced diffraction patterns that were blurred, indicating the lack of substantial long-range order in the crystals. Coexistence between P2 and C222 crystal forms has characteristics distinct from those previously seen in P1 and P2 coexistence. Domains of C222 or P2 in coexistence with P1 (in intermediate morphologies at pH 4.5-5.7) tend to be smaller, narrower, and more interspersed with P1 crystals than the coexisting C222/P2 domains observed in dendritic X’s at pH 6-7. In addition, although P1 domains are readily identified from C222 or P2 domains, C222 and P2 domains are difficult to distinguish from each other. In fact, even with diffraction analysis, we obtain patterns which are intermediate between P2 and C222, and these regions have varying degrees of P2 and C222 characteristics (Figure 4B). This observation suggests that there may be twinning or frequent alternations between C222 and P2 rows in these regions. For intermediate morphologies at pH 4.5-5.7 and dendritic X’s at pH 5.7-7, coexistence ratios do not depend on the region observed in the 2D domains; average ratios seen in the center, edges, or ends of a particular crystal are similar. However, variability in individual ratios within a crystal occurs. These deviations are likely due to local fluctuations, and large ratio differences can appear even in immediately adjacent regions within the crystals. Another source of variability may be the length of time required for crystal growth. It is possible that over time
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Figure 4. Diffraction patterns calculated from regions 0.4 µm apart (∼0.014 µm2 each) in a dendritic-X crystal at pH 6.75. (A) The diffraction pattern resulting from Fourier analysis gives lattice parameters consistent with the C222 crystal form. Reciprocal lattice vectors, a* and b*, are indicated. (B) The diffraction pattern resulting from Fourier analysis of an area adjacent to the region in part (A) results in an “amalgam” diffraction. The spacings between the reflections suggest that this result is not simply a superposition of the P2 and C222 diffractions but appears to be an intermediate form.
Figure 3. Crystal coexistence in H-shaped domains at pH 5.25. (A) The electron micrograph shows both P1 and C222 crystalline regions. P2 regions are also found in this particular crystal but are not shown in this micrograph. P1, P1 region; C222, C222 region. (Magnification ) ∼36 000×; Scale bar ) 50 nm.) (B) The diffraction pattern resulting from Fourier analysis of the indicated P1 region from part A gives lattice parameters consistent with the P1 crystal form. Reciprocal lattice vectors, a* and b*, are indicated. (C) The diffraction pattern resulting from Fourier analysis of the indicated C222 region from part A gives lattice parameters consistent with the C222 crystal form. Reciprocal lattice vectors, a* and b*, are indicated. (D) The diffraction pattern resulting from Fourier analysis of a crystalline region on this same crystal showing lattice parameters consistent with the P2 crystal form. Reciprocal lattice vectors, a* and b*, are indicated.
not only does a crystal grow larger but it may slightly favor one crystal form over another. Testing this phenomena is not straightforward because quantifying crystal ratios requires termination of the experiment, and crystal growth may vary slightly from one experiment to another depending on factors such as room temperature or protein injection rate. Because pH 5-6 is near the isoelectric point,3,4 electrostatic energy differences in this pH range may be minimal between the P1 and P2 crystal forms. The prevalence of C222 and P2 crystals and the absence of P1 crystals at high pH can be explained by an analysis of electrostatic charge densities around the streptavidin molecule at different pH values. Leckband35 found that at pH 7.2 the molecule primarily has a negative charge density on the surface, whereas at pH 5.9, a more balanced distribution of positive and negative charges exists. Because packing in the P1 configuration is denser than that in the C222 or P2 crystal forms, the close contact of streptavidin molecules at high pH would be prevented by repulsive interactions. Orientation of Biotin-Binding Sites and Preferred Growth Directions. Our crystal transfer methods and preferred growth direction determination are discussed in the Methods and Materials section and in Figure 1. Two-dimensional streptavidin crystals have a distinct (35) Leckband, D. E. Adv. Biophys. 1997, 34, 173.
Molecular Arrangement in 2D Streptavidin Crystals
orientation relative to the TEM grid to which they are bound. For the three types of crystals (P1, P2, and C222), we determined the molecular arrangement and orientation relative to the crystal morphology. This information will be useful for future 2D crystallization experiments in which streptavidin is mutagenized to determine the specific interactions important for crystal growth. The effects of differences in the protein-protein interactions can be observed in crystals with C222 symmetry. Streptavidin molecules which bind to a biotinylated lipid monolayer result in rectangular or H-shaped crystals with aspect ratios of 2-3.26,36 This anisotropy can be attributed to conformational changes upon biotin binding by streptavidin in two of the four subunits1 because such changes are near the crystal-contact region.34 Proteins bound to the monolayer by metal chelation of surface histidines do not undergo this conformational change, and the resulting crystal domains are square.16 Crystals with P1 Symmetry. We studied the anisotropy of needlelike morphologies comprising crystals with P1 symmetry (pH 4) (Figure 5). By comparing intensities and locations of individual reflections in our P1 diffraction (Figure 5B) to that reported in Hemming et al.,25 our experiments show the faster growth direction in this crystal form as drawn in Figure 5C. Because streptavidin tetramers are tightly packed, the preferred growth direction contains interactions both between biotin-bound subunits and between biotin-free subunits. Although an alternate indexing scheme exists, resulting in unit-cell parameters close to P1 values and a different molecular model, experimental evidence confirms the model we present in Figure 5. The angle between the reciprocal lattice vectors in our model is a value within experimental error of the reported value of 113°,25 and the alternate model is ∼5% higher. We also observe distinct lines in micrographs of P1 crystals, which correspond to the rows made by end-to-end molecules. Molecular modeling of the P1 lattice with space-filled streptavidin molecules gives empty space between molecules in adjacent rows, making these rows more distinct. Geometric analysis of the unit cell predicts a 50° angle between the preferred growth direction and these lines, and data from electron microscopy images coincide with this value. Finally, experiments using mutated proteins which incorporate additional interactions in the direction along these rows (in crystals with P1 symmetry) give morphologies which also confirm this model.37 Crystals with P2 Symmetry. In our previous work, we found a crystal form at intermediate pH values and determined that it shows P2 symmetry (because the oblique unit cell contains a 2-fold axis of rotational symmetry).14,38 The preferred growth and orientation of dendritic-X domains with this crystal type near pH 6 were difficult to obtain. Because of the low fluorescence efficiency of fluorescein near this pH, crystalline domains which transferred onto the TEM grids were difficult to identify, and after numerous attempts to obtain photographic documentation of crystalline domains on these grids, only one sample was appropriate for analysis. Determination of preferred growth and orientation in P2 crystals therefore was performed in H-shaped morphologies at lower pH values which contained high P2/P1 coexistence ratios (Figure 2D). We found the preferred growth direction to be along the subunits in the biotin(36) Ku, A. C.-T. The Growth of Two-Dimensional Streptavidin Crystals; Stanford University: Stanford, 1996. (37) Wang, S.-W.; Robertson, C. R.; Gast, A. P. 1999, in preparation. (38) Misell, D. L.; Brown, E. B. Electron Diffraction: An Introduction for Biologists; Elsevier: Amsterdam, 1987; Vol. 12.
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Figure 5. Molecular orientation of P1 crystals relative to needlelike domains. Lines with arrows at both ends indicate the direction of growth anisotropy. (A) Needlelike crystals (pH 4) and the corresponding preferred growth direction are shown. (B) Reciprocal lattice vectors, a* and b*, are defined as shown in this P1 diffraction pattern. (C) The molecular arrangement corresponding to part B is shown. Preferred growth direction has both biotin-free and biotin-bound streptavidin subunits in contact. Unit cell parameters, A and B, are indicated.
free direction (Figure 6). Comparison of this conclusion with the result of the previously mentioned dendritic-X, P2 crystal was performed, and this result was consistent with results obtained in the H-shaped domains. Crystals with C222 Symmetry. For dendritic-X and H-shaped crystals with C222 symmetry existing above pH 7, we found the preferred growth direction was faster along the biotin-bound subunits of streptavidin (Figure 7). This result is contrary to that reported in an earlier study,13 which incorrectly assigned the faster growth to the biotin-free direction. This discrepancy is most likely caused by the intricate nature of the data acquisition and image processing; at each step, the image is rotated or inverted, and an error in documentation of one part can give a different final result. In these experiments and analyses, we used a reference in each step to keep record of molecular orientation (as described in the Methods section). Molecular Arrangement in Coexisting Domains. The preferred growth direction and molecular orientation were also determined in coexisting crystals, and these results were consistent with data obtained from morphologies containing P1, P2, and C222 alone. Figure 8A
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Figure 6. Molecular orientation of P2 crystals relative to dendritic-X domains. Lines with arrows at both ends indicate the direction of growth anisotropy. (A) Dendritic-X crystals (pH 6) and the corresponding preferred growth direction are shown. (B) Reciprocal lattice vectors, a* and b*, are defined as shown in this P2 diffraction pattern. (C) The molecular arrangement for the P2 crystal form is shown. Preferred growth direction is along the biotin-free streptavidin subunits, indicated by open circles. Unit-cell parameters, A and B, are indicated.
shows the molecular arrangement of the three possible combinations of crystal coexistence: P1/P2, P1/C222, and P2/C222. Figure 8B is a TEM micrograph from an area containing coexisting P1 and C222 regions and shows that the model fits well with experimental evidence. Molecules in the C222 domain appear oriented in the same direction as proposed by our model, and observed adjacent rows in the P1 domains also correspond to the rows of molecules marked in Figure 8A. Some observations in this model are notable. The arrangement of the C222 and P2 crystal phases are such that the interfaces between the crystal types are in registry; distances between molecules in the preferred growth direction for both crystal forms are 58 Å apart. Our results also indicate that C222 and P2 molecules are rotated approximately 90° relative to each other when C222 and P2 coexistence is present, which enables the preferred growth direction for C222 to be in the biotin-
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Figure 7. Molecular orientation of C222 crystals relative to dendritic-X domains. Lines with arrows at both ends indicate the direction of growth anisotropy. (A) A dendritic-X crystal (pH 7) and the corresponding preferred growth direction are shown. (B) Reciprocal lattice vectors, a* and b*, can be defined as shown in this C222 diffraction pattern. (C) The molecular arrangement for the C222 crystal form is shown. Preferred growth direction is along the biotin-bound streptavidin subunits, indicated by filled circles. Unit-cell parameters, A and B, are indicated.
bound direction and the P2 crystal form to grow faster in the biotin-free direction. In addition, the P2 crystal has three of the four intermolecular interactions seen in the C222 crystal,14 showing that the energetics due to the loss of the fourth C222 contact is enough to change the preferred growth direction from biotin-bound to biotinunbound. Interfaces between P2 and C222 are reminiscent of both striations found in alloys and twinning,39 and the fact that intermediate “amalgam” diffraction patterns are seen (Figure 4B) also suggests that rows of molecules can switch between P2 or C222 lattices easily and often. These observations indicate that at the pH where such coexistence occurs (∼6.2 < pH < ∼6.8) interactions between biotin-bound and biotin-free subunits are weak, and that energetic differences between one configuration and the other (P2 or C222) are small. Crystals displaying coexistence are fragile and often fracture along the longest dimension of the crystal domain when perturbed. This direction coincides with the preferred growth direction at the molecular level and the (39) Olson, G. B., Owen, W. S., Eds. Martensite; ASM International: Materials Park, OH, 1992.
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Figure 8. Molecular arrangement of P1, P2, and C222 coexisting domains. (A) The preferred growth direction is indicated by the line with arrowheads at both ends. Lines with one arrowhead each indicate the direction in which visually observed adjacent rows in P1 domains are seen, corresponding to rows of molecules in the end-to-end position. (Scale bar ) 5 nm). (B) An area with coexisting P1 and C222 domains is shown. The adjacent arrows indicate the direction in which visually observed adjacent rows in P1 domains are observed, as mentioned in part A. (Scale bar ) 25 nm).
interfacial lines between the different crystal types. The observation of fragility in this same direction can be explained by either weaker interactions between coexisting adjacent crystal forms or one particular crystal phase exhibiting weaker overall interactions than the others. We observed that needlelike crystals with P1 symmetry give diffraction patterns which are less ordered than P1 crystals in coexistence with another crystal phase. It appears that the presence of P2 or C222 stabilizes P1 crystals, although the reasons for this are unclear. Analogy to Martensite Transformations. The transition from a crystal of higher to lower symmetry is characteristic of a class of structural changes called Martensitic transformations.39 In many important materials such as shape-memory alloys (Ni-Ti), this type of structural transformation occurs upon strain. The symmetry change from C222 to P1 after a change in pH is reminiscent of this type of transition, and the apparent hindrance to reverse transitions26 may be analogous to shape-memory effects. In addition, the striations observed in coexisting phases are similar to the twinned structures in Martensitic alloys. We are not the first to note the potential similarity between Martensitic transformations and 2D protein crystal transitions. Olson and Hartman40 discussed the structural changes occurring in the 2D crystals comprising the tail sheath of a bacteriophage following its penetration of a bacterium. It will be (40) Olson, G. B.; Hartman, H. J. Phys. 1982, C4, 855.
interesting in the future to monitor surface pressure or to attempt pressure jumps to test the Martensitic nature of the structural transitions occurring in 2D streptavidin as a function of pH. Chirality of Inverse-S Morphologies. Crystals which are entirely P1 display needlelike morphologies, but P1 crystals which contain frequent line defects or are interspersed with P2 crystals at a low percentage are chiral in shape. It appears that the asymmetry of the P2 arrangement and the streptavidin molecule (because of biotin binding) introduces a disruption to an otherwise achiral, needlelike morphology, which causes the “righthanded” curvature to exist. Although the asymmetry in the P2 interactions may be subtle and weak, it appears to have a profound effect on the morphology. For these chiral inverse-S crystal domains at the molecular level, the long, thin P2 domains and line defects coincide with the direction in which the crystal is growing at that particular point. In other words, the molecular arrangement is such that the preferred growth direction for a crystal with curvature is tangent to the curvature. This result supports the previous observation that polarization changes in the epifluorescent microscope give continuous intensity variations along the chiral domains.14 Conclusions In this study, we determined that various morphologies associated with the intermediate pH range of 4.5-6.5
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consist of different coexisting crystal ratios at the molecular level. Morphologies near pH 4.5 are primarily P1, but morphologies at increasingly higher pH values (up to pH ∼6) consist of crystals with increasing amounts of P2 and, consequently, higher P2/P1 ratios than those of crystals at lower pH values. Morphologies near pH 5.7 also comprise all three phases (C222, P1, and P2). Although domains at pH 6-7 appear to display the same dendritic-X morphology, these crystals actually consist of varying ratios of P2/C222, with domains closer to pH 7 containing higher amounts of C222. We also determined molecular arrangement and orientation relative to crystal phases and morphologies. As pH is changed, surface amino acids change their electrostatic characteristics, resulting in different proteinprotein interactions and consequently different crystal forms. Streptavidin molecules with P1 arrangement show strong anisotropy and appear to preferentially grow in a direction which contains both interactions between biotinfree subunits and interactions between biotin-bound subunits. In P2 crystals, the preferred direction is along biotin-free subunit monomers, whereas in C222 crystals, it is along the biotin-bound monomers. This information, together with crystal packing data and molecular model-
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ing, allows for the identification of potential key interactions and contact regions. Through mutagenesis, specific amino acids can be replaced to alter the attraction or repulsion between contact regions to obtain modified crystal properties. In our current work, for example, we have modified an interaction in the P1 crystal form, resulting in interesting morphological, crystallographic, and thermodynamic changes in both P1 and P2 crystals.37 Such investigations show that molecular information is significant in the study of interactions which exist in 2D protein crystals and which govern their growth. The manipulation of intermolecular interactions which lead to the control of protein array properties will help further the development of technologies using protein monolayers. Acknowledgment. We thank Roger Kornberg for graciously allowing us the use of his laboratory facilities, Lynne Mercer for assistance with the TEM, and Claudia Poglitsch for helpful discussions. This work was supported by NSF Grant BCS-9202220 to A.P.G. and C.R.R. and a Whitaker Foundation Graduate Fellowship to S.W.W. LA981038G
