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Influence of pH on Two-Dimensional Streptavidin Crystals Michael T. Yatcilla, Channing R. Robertson, and Alice P. Gast* Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025 Received March 3, 1997. In Final Form: November 5, 1997 To obtain a general understanding of the effect of intermolecular interactions on the mechanisms of two-dimensional protein crystallization, we grow protein crystals and elicit a bulk molecular manipulation by changing system pH. Two-dimensional crystals of the bacterial protein streptavidin grown on a biotinylated lipid monolayer at an air-water interface, in the presence of the noncrystallizable impurity avidin, exhibit crystallographic and morphological changes as a function of subphase pH. Large twodimensional crystalline arrays form within minutes across a pH range from 1.5 to 11. Crystals exhibit different pH-dependent structures, lattices with P1 symmetry for 1.5 < pH < 5, P1 and P2 lattices for 5 < pH < 6, and C222 lattices for 7 < pH < 11. P1 crystals nucleate rapidly and form thin needle-shaped crystals consistent with a strong growth anisotropy between the two crystallographic growth directions. C222 crystals grow more isotropically and exhibit H- and X-shapes. The nucleation rates and aspect ratios of C222 crystals are also pH-dependent, both properties increasing with increasing pH. The transition from C222 to P1 or P2 crystals can be accomplished in minutes by lowering the system pH. The reverse transition, however, does not occur subsequent to a corresponding increase in system pH. Instead, new C222 crystals form, but no reconfiguration of existing crystals is observed.
Introduction Proteins assemble into ordered arrays exactly onemolecule thick under a variety of conditions. These arrays have been found on air-water interfaces,1 at air-mercury interfaces,2 and, most commonly, on lipid monolayers at air-water interfaces.3 Such arrays are useful in protein structure determination,4 as the complete three-dimensional structure of proteins can be obtained from twodimensional protein crystals. The two-dimensional5 approach is preferable both in situations where proteins are recalcitrant to three-dimensional crystallization and also in situations where protein is scarce, as high-resolution 2D lipid-layer crystals require only micrograms of protein. In addition, two-dimensional crystals can serve as templates for epitaxial growth of three-dimensional protein crystals.6 Finally, the protein is oriented with an entire face unconstrained by crystal. Proteins with an active site can be crystallized in “active” or “passive” states by the presence or absence of the protein’s substrate in the subphase. Lipid-layer crystallization is of particular utility, as it permits one to exploit natural protein ligation behavior to confine the protein to the lipid layer. Several proteins have been crystallized when bound to either synthetic or natural lipid ligands.7 In addition, proteins have been crystallized on lipid layers through inherent affinity for * Corresponding author. (1) Yoshimura, H.; Scheybani, T.; Baumeister, W.; Nagayama, K. Langmuir 1994, 10, 3290. (2) Nagayama, K. Mater Sci. Eng. 1994, C1, 87. (3) Reviews: Kornberg, R. D.; Darst, S. A. Curr. Opin. Struct. Biol. 1991, 1, 642. Engel, A.; Hoenger, A.; Hefti, A.; Henn, C.; Ford, R. C.; Kistler, J.; Zulauf, M. J. Struct. Biol. 1992, 109, 219. Brisson, A.; Olofsson, A.; Ringler, P.; Schmutz, M.; Stoylova, S. Biol. Cell 1994, 80, 221. (4) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.; Blankenburg, E. W.; Ribi, H. O.; Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991, 59, 387. (5) Abbreviations used in this paper: 2D, two-dimensional; TEM, transmission electron microscopy; biotin-DHPE, N-(6-((biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; DOPC, 1,2-dioeoylphospatidylcholine; FITC, fluorescene isothiocyanate. (6) Edwards, A. M.; Darst, S. A.; Hemming, S. A.; Li, Y.; Kornberg, R. D. Struct. Biol. 1994, 1, 195.
positively charged lipids8 as well as metal-chelated ones.9 The proteins can also be fluorescently labeled, and the resulting two-dimensional morphologies and growth behavior can be observed with a fluorescence microscope in a method analogous to that used in the study of twodimensional lipid domains in the last decade.10 We seek a general understanding of the crystallization process in two dimensions, from a molecular as well as macroscopic standpoint. To accomplish this, we observe large (greater than 5 µm) crystals as they grow on a Langmuir trough and examine molecular features of the crystals using transmission electron microscopy (TEM). As our model system, we choose the well-studied streptavidin-biotin system, which crystallizes readily beneath preformed biotinylated lipid monolayers.11 These protein crystals can be observed with a fluorescence microscope.12 The homologous protein avidin can be introduced to the system as a noncrystallizable impurity with a similar affinity for biotin. Avidin dilutes the 2D crystals and permits streptavidin crystals to exhibit an advanced development of morphological features.13 Fluorescence microscopy of these features is a noninvasive means of observing nucleation and growth on a large scale, with morphological development yielding quantitative infor(7) Annexin-V.; Mosser, G.; Ravanat, C.; Freyssinet, J.-M.; Brisson, A. J. Mol. Biol. 1991, 217, 241. Voges, D.; Berendes, R.; Burger, A.; Demange, P.; Baumeister, W.; Huber, R. J. Mol. Biol. 1994, 238, 199. Cholera Toxin B-subunit: Ribi, H. O.; Ludwig, D. S.; Mercer, K. L.; Schoolnik, G. K.; Kornberg, R. D. Science 1988, 239, 1272. Mosser, G.; Brisson, A. J. Struct. Biol. 1991, 106, 191. (8) RNA Polymerase II: Darst, S. A.; Kubalek, E. W.; Edwards, A. M.; Kornberg, R. D. J. Mol. Biol. 1991, 221, 347. (9) Shnek, D. R.; Pack, D. W.; Sasaki, D. Y.; Arnold, F. H. Langmuir 1994, 10, 2382. (10) Mohwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441. McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171. Knobler, C. M. Annu. Rev. Phys. Chem. 1992, 43, 207. (11) Kornberg, R. D. Formation of two-dimensional crystals of proteins on lipid layers. In UCLA Symposia On Molecular And Cellular Biology New Series; Oxender, D. L., Ed.; Liss: New York, 1987; Vol. 69, p 175. (12) Blankenburg, R.; Meller, P.; Ringsdorf, H.; Salesse, C. Biochemistry 1989, 28, 8214. More recently in: Frey, W.; Schief, W. R.; Vogel, V. Langmuir 1996, 12, 1312. (13) Ku, A. C.; Darst, S. A.; Kornberg, R. D.; Roberston, C. R.; Gast, A. P. Langmuir 1992, 8, 2357. Ku, A. C.; Darst, S. A.; Robertson, C. R.; Gast, A. P.; Kornberg, R. D. J. Phys. Chem. 1993, 97, 3013.
S0743-7463(97)00237-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/01/1998
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mation about growth kinetics and the intermolecular interactions that drive crystallization. Streptavidin crystals typically form large-scale morphologies with a pronounced growth anisotropy.12,13 This arises from the nature of the protein binding to the biotinylated lipid layer. The structural difference on binding biotin results in two distinct pairs of contacts, one being more energetically favored than the other.14 Recent work on crystallizing streptavidin with nonbiotinylated lipids demonstrates that this anisotropy does not occur when the pairs of contacts are identical.15 We wish to exploit the fact that streptavidin crystallizes in multiple forms.16 We manipulate our system between these crystal forms to observe the macroscopic effects with fluorescence microscopy. The existence of these multiple crystal forms, with different sets of intermolecular contacts, affords the opportunity to examine different disorderto-order phase transitions. Additionally, we can attempt to observe microscopic changes, morphological changes, and reversibility during a phase transition between wellordered phases. We can do this readily with the streptavidin system by manipulating the subphase pH under preformed crystals. Materials All proteins and lipids were used directly as purchased. Streptavidin from Streptomyces avidinii was obtained from Boehringer Mannheim (Indianapolis, IN). Avidin was obtained from Sigma (St. Louis, MO). Both were stored at a concentration of 2 mg/mL in 0.5 M NaCl, 50 mM Na2H2PO4 (the crystallization buffer) at pH 7.5 at -20 °C prior to experiments. N-(6-((Biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Biotin-DHPE) was obtained from Molecular Probes (Eugene, OR). DOPC was obtained from Avanti Polar Lipids. Both lipids were stored in 50/50 chloroform/hexane at -20 °C prior to experiments. Fluorescene isothiocyanate (FITC) was obtained from Sigma. Streptavidin was labeled with FITC by mixing the dye and the protein at a ratio of 5 molecules of FITC to 1 molecule of streptavidin. The dye was allowed to react with the protein for 8 h at 25 °C. Excess dye was removed from the protein using a Sephadex 50 (Sigma) column. The protein concentration was determined from absorbance measurements (492 nm) using an HP spectrophotometer with BSA standards.
Experimental Section Two-dimensional crystals of streptavidin were grown in a Delrin Langmuir trough (50 × 20 × 5 mm deep) with a Teflon barrier. The subphase buffer (see above) at the desired pH was filtered with a 0.2 µm filter (Millipore) and placed in the trough. The trough was then cleaned and verified to be free of surfaceactive impurities by the absence of a surface-pressure change following barrier compression. A lipid mixture of 1:10 Biotin-DHPE/DOPC at a concentration of 0.17 mg/mL was used in all experiments. Approximately 4 µL of this lipid mixture was pipetted onto the trough at the airbuffer interface in several small drops across the surface of the trough. When the solvent evaporated, the barrier was moved across the trough surface, compressing the lipid monolayer to a surface pressure of 25 dyn/cm. A mixture of 2 mg/mL streptavidin and avidin solution was slowly injected with a 25-µL syringe into the subphase under the lipid layer. The injection was made over a period of 1 min to avoid convection in the lipid layer. For these experiments, a (14) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85. (15) Frey, W.; Schief, W. R., Jr.; Pack, D. W.; Chen, C.-T.; Chilkoti, A.; Stayton, P.; Vogel, V.; Arnold, F. A. Proc. Natl. Acad. Sci. USA 1996, 93, 4937. (16) 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.
Yatcilla et al. ratio of 3:1 avidin/FITC-streptavidin was used; the total protein concentration was 20 µg/mL. Crystals were observed with a Zeiss Axioplan epifluorescence microscope over a period of hours and photographed with a 35mm camera. For experiments examining the transition between one crystal form and another, the experiments were set up as described. After several hours, when crystal growth had stopped or slowed to an imperceptible rate, 0.1 M or 1.0 M NaOH or HCl was slowly introduced to the subphase in a manner analagous to protein injection, to bring the system to a desired overall pH. Crystals were transferred to TEM grids by touching carboncoated Cu-Rh “finder” grids (mesh size 400sElectron Microscopy Science, Fort Washington, PA) to the lipid surface. The grids were rinsed with 5 µL of filtered, deionized water for 15 s, dried with filter paper, and then stained with 5 µL of filtered 1.0% uranyl acetate solution for 1 min. The grids were again dried with filter paper and stored in a desiccator prior to electron microscopy. Subsequent TEM, imaging, and analysis are described in detail in ref 4.
Results Crystals grew abundantly for 1.5 < pH < 11. The time between the start of an experiment and the time when the crystals were large enough to be observed with the epifluorescence microscope (approximately 5 µm) varied among experiments but typically was on the order of 1-5 min. For pH > 7.0, we found the expected C222 crystals (see Figure 1a). These crystals have a square lattice (γ ) 90°), with a ) b ) 58 Å. For pH < 5.0, we found only P1 crystals (Figure 1b); these crystals have a nonsquare lattice (γ ) 113°) and more a compact structure (a ) 58 Å, b ) 50 Å). For 5.0 < pH < 6.0, a combination of crystal forms was found, including a new third crystal form with P2 symmetry, discussed in more detail elsewhere.17 When viewed under the fluorescence microscope, the low-pH crystals (P1) have morphologies with large aspect ratios (see Figure 2). At early times (Figure 2a), the crystals are very long and thin and nucleate rapidly. At later times, the crystals quickly crowd into each other, inhibiting further growth. Only when the crystals have no further room to grow along their length do they eventually thicken, ultimately resembling “cigar” shapes (Figure 2b). Near pH 5, nucleation rate decreases, and larger cigar-shaped P1 crystals are observed.17 For experiments performed above pH 7, C222 crystals are observed exclusively. They exhibit “H”- and “X”shapes, characteristic of a noncrystallizable impurity,12 in this case, the homologous protein avidin. In all cases, there is a pronounced anisotropy, with an average crystal aspect ratio (as defined by length of long edge/length of short edge) of approximately 2. Figure 3 shows a variety of C222 crystals imaged with the fluorescence microscope. At increasing pH values, there is a larger number of smaller crystals (Figure 3b). The higher nucleation rate retards the morphological development, and crystals at high pH rarely evolve beyond a rectangular or “H”-shape (Figure 3c). At still higher pH, these trends of rapid nucleation and resulting crowding continue. Figure 4 shows a large number of rectangular nuclei which eventually merge into each other. This increased nucleation rate results in a “tiling” effect that yields a much higher surface coverage of crystal than in the lower pH case (e.g., Figure 3a). To observe the transitions between these two crystal forms, we performed several sets of experiments on the preformed crystals. In these experiments, we find that C222 crystals readily break up and reform as P1 crystals following a reduction in pH. Large, rectangular “H”- or (17) Wang, S.-W.; Poglitsch, C.; Yatcilla, M. T.; Roberston, C. R.; Gast, A. P. Langmuir 1997, 13, 5794.
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Figure 1. Two-dimensional crystal forms of streptavidin. Amino acid backbones of streptavidin tetramers as viewed from above are shown. (a) C222 crystals,11 a ) b ) 58 Å, γ ) 90°. In this work, this crystal form is observed exclusively above pH 7.0. Van der Waals radii of biotin molecules are also shown. (b) P1 crystals,16 a ) 58 Å, b ) 50 Å, γ ) 113°. P1 crystals are observed below pH 5.0. For 5.0 < pH < 6.0, the P1 crystal form coexists with a new crystal form not shown here.17
“X”-shaped crystals grown at high pH (7 < pH < 10) change into “sliver” shapes when the system pH is lowered (to 3 < pH < 5). Figure 5 shows an example of this transition. The large “X”-shaped crystals appear to splinter into small slivers from the exterior inward. Initial slivers are observed at the perimeter of the large crystals within 5 min of a pH change, with complete replacement of the C222 crystals by P1 crystals within hours. When the pH
is reduced to 1.0 or less, the crystals dissolve entirely, disappearing from the exterior inward. When P1 crystals grown at pH 5 have their pH raised to 8, nucleation of rectangular “H”-shapes is observed at the edges of the crystals as well as in the bulk (see Figure 6). These new crystals exhibit C222 symmetry. The original P1 crystals remain unchanged. P1 crystals grown at 2 < pH < 4 do not change to C222
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Figure 2. Epifluorescence micrographs of P1 crystals. The scale bar is 100 µm. The system is 1:3 FITC-streptavidin/unlabeled avidin at pH 4.0, with total protein concentration 20 µg/mL. (a) Five minutes after injection of protein, the crystals are very long and thin. (b) Sixty minutes after injection of protein, the crystals have thickened.
Figure 3. Epifluorescence photographs of C222 crystals at different pH, for the same system as in Figure 2. The scale bar is 100 µm. (a) At pH 7.0, “X”-shaped crystals are formed with aspect ratio ∼2.0. (b) At pH 8.0, “H”-shaped crystals are formed with aspect ratio ∼2.0. There are a larger number of nuclei, and crystals have become sterically hindered from growing into large “X”-shapes as in part a. (c) At pH 9.0, crystals are approaching rectangular shapes with aspect ratio ∼2.5.
crystals following an increase in pH. Crystals that incubate at pH 4 (e.g., Figure 2b) for hours and are subsequently raised to pH 7-10 do not exhibit any morphological changes over days. Raising the pH to 11 or above resulted in rapid dissolution of all crystals.
Discussion The anisotropy present in all macroscopic morphologies results from the geometry of the streptavidin tetramer bound to a biotinylated lipid layer. In these experiments,
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Figure 4. Epifluorescence micrographs of pH 10 crystals, for the same system as in Figure 2. Scale bar is 100 µm. (a) At 5 min after injection, a very large number of small crystals with aspect ratio ∼2.5 become apparent. (b) At 20 min after injection, crystals have crowded.
Figure 5. Epifluorescence micrograph of C222 crystal in the midst of a transition to a P1 crystal at pH 4. Photograph taken 10 min after the system pH was changed. The small, slivershaped crystals with P1 packing eventually completely replace the large C222 dendritic crystals.
streptavidin crystallizes with two of the four biotin binding sites bound to biotin and facing the lipid monolayer with the other two biotin-binding sites unbound and facing down (toward the aqueous subphase). In the case of the C222 crystal form, as shown in Figure 7, there are only two pairs of contacts between adjacent molecules of streptavidin in the crystal: the biotin-bound/biotin-bound subunit pair (Figure 7a) and an unbound/unbound subunit pair (Figure 7b). These two sets of contacts are not identical, as the stretch of amino acid residues numbered 46-50 undergoes a conformational change with biotin binding and moves into the interaction region in the unbound case (Figure 7b). From the molecular models in
Figure 6. Epifluorescence micrograph of coexisting C222 and P1 crystals at pH 7. The lightest shades are sliver-shaped P1 crystals grown at pH 5. The darker and rectangular crystals are C222 crystals.
Figure 7, it is unclear whether this additional interaction is repulsive or attractive, but it has been shown that growth is faster in the direction of unbound contacts.13 It appears to be insensitive to pH change, however, as the aspect ratios observed across the pH range 7-11 remain approximately 2-2.5. There are other, more subtle conformational differences between biotin-bound and biotin-unbound streptavidin pairs which may also have an effect on the relative energies of the two pairs of contacts and in turn affect the growth anisotropies we observe.
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Figure 7. Pairs of biotin-bound and unbound streptavidins. This is the orientation and spacing of protein molecules in the C222 crystal form (as shown in Figure 1a). In C222 crystals, there are two of each type of interaction (a and b) per streptavidin tetramer. Each is associated with a growth direction. Amino acid backbones are shown, as well as Van der Waals radii for selected amino acids [residues 20 (Thr), 49 (Asn), and 100 (Ala)] and biotin. These amino acid residues make the closest approach to each other in neighboring molecules in C222 crystal forms. (a) For the biotin-bound/biotin-bound interaction the “arm” associated with biotin binding is holding the biotin in place.13 As a result, the arms (residues 46-50) are over 10 Å from each other. (b) For the unbound/ unbound interaction, the arms face toward each other and introduce another set of contacts.
For P1 crystals, the anisotropy is even more pronounced. At early times (Figure 2a), when the crystals grow unhindered by neighboring crystals, the resultant morphology is highly anisotropic, the crystal shapes being best described as “lines”. It appears that one of the two growth directions is highly preferred, and the crystal begins to grow substantially in the second direction (Figure 2b) only when constrained by neighboring crystals. This difference in anisotropy may be related to the obvious differences between the P1 crystal (Figure 1b) and the C222 crystal just discussed. First, in the P1 crystal the two growth directions are not orthogonal to each other, and growth anisotropy between the two directions on the molecular level is amplified on the macroscopic level. Second, there are more than two sets of contacts in the P1 crystal form, each of which contributes to the relative energetics of adding a molecule to the lattice in either position. This makes a direct comparison of the two growth directions difficult on a molecular scale and thus renders the macroscopic information of relative growth rates potentially more useful. The experiments with abrupt pH changes indicate reversibility in one direction only. Transitions from C222
to P1 lattices occur readily, while the reverse transitions, P1 to C222, are not observed. Under conditions where there is a small number of large crystals (as shown in Figure 6) however, we do see the appearance of new C222 crystals. This suggests that protein previously adsorbed to the lipid layer but not incorporated into crystal grows into C222 crystals, but protein existing in the P1 crystal state does not change into the C222 form. We note that the P1 lattice has a much more compact packing of the streptavidin tetramers (see Figure 1b): the C222 form requires an additional 464 Å2 of monolayer area per streptavidin tetramer. It is possible that this monolayer area is not available to the P1 crystal, and hence the more compact lattice associated with the P1 form is inhibited from expanding into the more open C222 form. In addition, the extra interprotein contacts present in the P1 crystal may be strong contacts which form a substantial energy well, effectively trapping the system in a metastable state. The nucleation rate is also highly pH-sensitive. The increase in number of nuclei and corresponding steric hindrance hampers advanced crystal morphological development (e.g., appearance of secondary dendrites) but
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also increases the relative amount of surface monolayer covered by crystal. If we compare Figure 3a (at pH 7) to Figure 4b (at pH 10), both C222 crystals, we see that, in the higher pH experiment, crystalline streptavidin covers a much larger percentage of the monolayer in a similar period of time. Growth of individual crystals appears to take place at roughly the same rate, however, whether there are many or few crystals visible on the surface. Conclusions A molecular manipulation of the streptavidin-biotin system can be readily effected using pH control. Multiple distinct crystal forms with a corresponding range of morphologies can be observed by varying the system pH. These crystal forms have different sets of intermolecular contacts, and the energetics of these contacts appear to be pH-sensitive. As a result of this sensitivity, 2D streptavidin crystals change their bulk crystal form at different pH values, often within a very small pH range. Additionally, the nucleation and growth behavior of the 2D crystal forms are quite sensitive to pH changes. That pH and electrolyte concentrations are important in forming crystals is well-known to protein crystallographers. Most typically, however, this sensitivity has been represented only in a very general and qualitative fashion, as in determining under what conditions crystals grow most rapidly.18 Here we make a detailed observation of the effect of pH on 2D crystallization and suggest the utility of controlled pH adjustment as a quantitative tool towards understanding the 2D crystal growth process. More specifically, the observed transition of crystals from C222 to P1 or P2 following a pH change suggests possible experiments and applications. Coupled with the overall efficiency of crystallization at high pH values, this ability to transform one crystal form to another might be used to exploit the very high rate of nucleation occurring at elevated pH. Crystal growth (at prescribed conditions) could be initiated in the rapid-nucleation form to “seed”
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a number of crystals and then changed abruptly into the slower nucleating form once sufficient nuclei are present. This appears to be a 2D analog of the seeded epitaxial growth of 3D crystals and may be a complementary technique in rapid growth of large crystals. In this work, we address fundamental driving forces of crystallization in two dimensions. With pH as the molecular manipulation, we visualize a number of different crystal forms as the molecular observation and a variety of morphologies and nucleation rates as the macroscopic observation. Other molecular manipulations, such as making point mutations19 to the tetramer, provide even finer control of the system energetics. Whichever method is used, we can apply the techniques described here to observe the interplay of altered intermolecular energetics and the resulting changes in such fundamental crystallization properties as crystal packing, nucleation rates, and growth behavior. Acknowledgment. Figures 1 and 7 were generated using RasMac v2.6, the Macintosh version of a molecularrendering program by Roger Sayle available for free download at ftp.dcs.ed.ac.uk/pub/rasmol. The PDB structure of the streptavidin tetramer with two biotin-bound subunits was generously provided by Professor Patrick Stayton of the University of Washington. M.T.Y. would like to thank Lynne Mercer, Claudia Poglitsch, and Szu Wang for assistance with TEM processing and Fidel Rebeles for experimental assistance. M.T.Y. is supported by a NIH Graduate Traineeship in Biotechnology (T32 GM08412) and a William K. Bowes Fellowship. This work is funded by NSF Grant BES-9202220. LA970237H (18) Cox, M. J.; Weber, P. C. J. Cryst. Growth 1988, 90, 318. (19) Sano, T.; Cantor, C. R. Proc. Natl. Acad. Sci. USA 1995, 92, 3180. Chilkoti, A.; Tan, P. H.; Stayton, P. S. Proc. Natl. Acad. Sci. USA 1995, 92, 1754.
