Molecular Basis for Ionic Strength Dependence and Crystal

DOI: 10.1021/la9801918. Publication Date (Web): July 30, 1998 ... Biotemplating of Metallic Nanoparticle Arrays Through Site-Specific Electrostatic Ad...
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Langmuir 1998, 14, 4683-4687

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Molecular Basis for Ionic Strength Dependence and Crystal Morphology in Two-Dimensional Streptavidin Crystallization Todd C. Edwards,† Sandy Koppenol,† Wolfgang Frey,† William R. Schief, Jr.,† Viola Vogel,† Ronald E. Stenkamp,‡ and Patrick S. Stayton*,† Department of Bioengineering, University of Washington, Seattle, Washington 98195-7962, and Department of Biological Structure and Biomolecular Structure Center, University of Washington, Seattle, Washington 98195-7742 Received February 17, 1998. In Final Form: April 21, 1998 The two-dimensional crystallization of streptavidin at functionalized lipid interfaces is one of the best studied model systems for investigating molecular self-assembly processes. This system also provides an opportunity to elucidate the relationship between protein-protein molecular recognition, crystallization solution conditions, and crystal properties such as coherence, space group symmetry, and morphology. A better understanding of these relationships may aid in the design of rational strategies for promoting high-quality protein crystallization and for controlling protein assembly at interfaces in the biomaterials and nanotechnology fields. Here we show that two-dimensional streptavidin crystallization is kinetically controlled and that formation of a single electrostatic interaction at the crystal contact interfaces is a key energetic determinant of the kinetic barriers controlling crystal morphology. Our results also demonstrate that this electrostatic interaction at the streptavidin protein-protein interfaces is responsible for the ionic strength dependence of streptavidin crystallization. Molecular modeling studies of the wild-type crystal that displays C222 symmetry suggested that the side-chain amines of lysine 132 from adjacent proteins interact with each other across the dyad-related crystal contacts. Leucine was substituted at this position (K132L) to remove the need for bridging counterions. Unlike wild-type streptavidin, the K132L mutant crystallizes with rectangular morphology on buffer or on a pure water subphase and analysis of the electron micrographs demonstrates that the crystal retains C222 symmetry in the presence or absence of salt. The kinetic barriers associated with formation of this electrostatic interaction thus underlie the wild-type butterfly crystal morphology.

Introduction Although high-quality protein crystallization remains an important goal that often limits the application and resolution of X-ray crystallography, the fundamental energetic and molecular recognition processes controlling crystal nucleation, growth, morphology and coherence are poorly understood. The well-studied streptavidin twodimensional (2D) crystal model system provides an opportunity to investigate the relationship between protein-protein interactions at crystal contact interfaces and various crystallization parameters. Streptavidin assembles into 2D crystals with the crystallographic space group C222 under biotinylated lipid monolayers at neutral pH.1-3 This system has been extensively studied by a variety of spectroscopic and diffraction techniques, and the crystals have a butterfly or X-shaped morphology when imaged by Brewster angle or fluorescence microscopy.1-12 Because two of streptavidin’s subunits are positioned away * To whom correspondence may be addressed: Patrick S. Stayton, Box 357962, Department of Bioengineering, University of Washington, Seattle, WA 98195.; phone, (206) 685-8148; fax, (206) 6858256; e-mail, [email protected]. † Department of Bioengineering. ‡ Department of Biological Structure and Biomolecular Structure Center. (1) 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, 387-396. (2) 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, 308-316. (3) Avila-Sakar, A. J.; Chiu, W. Biophys. J. 1996, 70, 57-68. (4) Ahlers, M.; Blankenburg, R.; Grainger, D. W.; Meller, P.; Ringsdorf, H.; Salesse, C. Thin Solid Films 1989, 180, 93-99. (5) Blankenburg, R.; Meller, P.; Ringsdorf, H.; Salesse, C. Biochemistry 1989, 28, 8214-8221.

from the monolayer and exposed to the subphase, a second layer of biotinylated biomolecules can be ordered on the streptavidin template.13-16 The two-dimensional crystals have also been used as templates to promote threedimensional crystallization.17,18 Streptavidin forms 2D crystals with C222 symmetry under biotinylated lipids at neutral pH. A computer model of the contact was constructed based on a high-resolution 3D crystal with I4122 symmetry. The structure of the C222 crystals is the same as that found in a layer in the I4122 crystal, and thus this layer represents a good model of the protein-protein crystal contacts. The molecular symmetry of the streptavidin tetramer and the C222 2D (6) Kornberg, R. D.; Darst, S. A. Curr. Opin. Struct. Biol. 1991, 1, 642-646. (7) Schmidt, A.; Spinke, J.; Bayerl, T.; Sackmann, E.; Knoll, W. Biophys. J. 1992, 63, 1185-1192. (8) Furuno, T.; Sasabe, H. Biophys. J. 1993, 65, 1714-7. (9) Haas, H.; Mo¨hwald, H. Colloids Surf., B 1993, 1, 139-148. (10) Haas, H.; Mo¨hwald, H. Langmuir 1994, 10, 363-366. (11) Haas, H.; Brezesinski, G.; Mo¨hwald, H. Biophys. J. 1995, 68, 312-4. (12) Frey, W.; Schief, W. R.; Vogel, V. Langmuir 1996, 12, 13121320. (13) Herron, J. N.; Mu¨ller, W.; Paudler, M.; Riegler, H.; Ringsdorf, H.; Suci, P. A. Langmuir 1992, 8, 1413-1416. (14) Mu¨ller, W.; Ringsdorf, H.; Rump, E.; Wildburg, G.; Zhang, X.; Angermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 17061708. (15) Spinke, J.; Liley, M.; Guder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821-1825. (16) Fujita, K.; Kimura, S.; Imanishi, Y.; Rump, E.; van Esch, J.; Ringsdorf, H. J. Am. Chem. Soc. 1994, 116, 5479-5480. (17) Darst, S. A.; Edwards, A. M. Curr. Opin. Struct. Biol. 1995, 5, 640-4. (18) Edwards, A. M.; Darst, S. A.; Hemming, S. A.; Li, Y.; Kornberg, R. D. Nature, Struct. Biol. 1994, 1, 195-197.

S0743-7463(98)00191-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/30/1998

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crystal symmetry dictate that each subunit makes only one set of interprotein contacts, which are the same for each subunit to within crystallographic error. Our model of these crystal contacts suggested that lysine 132 interacts with its dyad-related counterpart across the proteinprotein interface, potentially giving rise to an unfavorable electrostatic interaction (Figure 1). Because 2D crystallization of streptavidin has always been performed at high ionic strength,5 we hypothesized that counterions such as chloride are necessary to shield the charges on the sidechain amines. To test this hypothesis, site-directed mutagenesis was used to change the lysine at residue 132 to leucine. Leucine was chosen to remove the charges and thus the need for counterions at the wild-type crystal contact, while potentially retaining hydrophobic/van der Waals interactions mediated by side-chain hydrocarbons. Recombinant wild-type (core streptavidin with residues 13-139) and K132L streptavidin were compared in crystallization experiments in purified water (resistivity ) 18 MΩ cm, pH 6.5) and in high ionic strength buffer (10 mM HEPES, 250 mM NaCl, 10 µM EDTA) at pH 7.8 and at pH 6.5 to control for pH-mediated effects. Quantitative Brewster angle microscopy (BAM)12,19,20 was used to observe the streptavidin crystallization process. Materials and Methods Molecular Modeling. Computer modeling was done on an O2 workstation (Silicon Graphics Computer Systems, Mountain View, CA) with the programs PSSHOW (Swanson, E: PSSHOW: Silicon Graphics 4D Version. Seattle, WA 1990) and XtalView.21 Atomic coordinates for a single streptavidin subunit were obtained from the Protein Data Bank as the file 1sld.pdb.22 Those coordinates were chosen because the two-dimensional C222 plane is represented in the 3D crystal of space group I4122, and thus has similar protein-protein contacts. The coordinates of the monomer were transformed to produce the entire tetramer, which was then copied and translated according to the published unit cell1,4,5 to model the protein-protein contact. Site-Directed Mutagenesis and Protein Chemistry. The K132L coding sequence was generated by cassette mutagenesis of the wild-type streptavidin gene. Synthetic sense and antisense oligonucleotides (Integrated DNA Technologies, Inc., Coralville, IA) were annealed to form the mutant cassette, and subsequently subcloned between the Mlu I and Pst I restriction sites in the streptavidin gene after removal of the complementary wild-type cassette. The K132L mutant gene was sequenced and then subcloned into the pET 21a(+) expression vector and transformed into BL21(DE3) E. coli cells for expression. Cells were grown in 2xYT medium until OD600 ) 0.6-1.0, at which point protein expression was induced with 0.5 M isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were harvested after 3 h. Isolated inclusion bodies were then denatured in solubilization buffer (50 mM Tris, 6 M guanidine HCl, pH 7.5) and refolded by slowly dripping into a stirred refolding solution (50 mM Tris, 100 mM NaCl, 5 mM EDTA, 0.1 mM PMSF, pH 7.5) at 4 °C. Refolded protein was concentrated by ultrafiltration and purified via iminobiotin affinity chromatography (Pierce, Rockford IL). The purified streptavidin mutants were compared to wild type by electrospray ionization mass spectrometry, which yielded a measured molecular mass of 13 254 Da vs 13 256 Da predicted by the primary sequence. Crystallization Studies. 2D crystallization was performed in a 6 mm deep Teflon Langmuir trough with three different subphases: 10 mM HEPES, 250 mM NaCl, 10 mM EDTA, pH 7.8 and pH 6.5, and Nanopure water, pH 6.5. The biotinylated lipids N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (B-DPPE), and N-((6-(biotinoyl)amino)hexanoyl)1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (B-xDPPE) (Molecular Probes, Eugene, OR) were spread from (19) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590-4592. (20) He´non, S.; Meunier, J. Rev. Sci. Instrum. 1991, 62, 936-939. (21) McRee, D. E. J. Mol. Graphics 1992, 10, 44-46. (22) Katz, B. A. Biochemistry 1995, 34, 15421-9.

Figure 1. Molecular model of the streptavidin C222 twodimensional unit cell. The model was generated from the C222 two-dimensional plane found in the three-dimensional I4122 crystal. (a, top) The streptavidin backbone is represented in ribbon format and the lysine 132 side chains are shown as a ball-and-stick representation at the protein-protein contacts. Individual monomers are colored differently in each monomer. The C-terminal residues 134-139 are absent because they are not visible in the 3D crystallographic electron density maps. (b, bottom) Side view of the lysine 132 interaction. Protein subunits that are not involved in this contact have been omitted for clarity The side-chain amines are approximately 3.5 Å apart in this model. Both figures were created with Molscript (Kraulis, P. J. J. Appl. Crystallogr. 1991, 24, 946-950.). chloroform to a calculated surface pressure of 1-2 or 26-28 mN/m, respectively. After allowing time for chloroform evaporation, the protein was injected into the subphase to yield concentrations of either 19, 38, or 77 nM. All protein solutions used for crystallization experiments were dialyzed against water

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Figure 2. Brewster angle microscopy images of wild-type and K132L streptavidin crystals. (a) Wild-type recombinant streptavidin forms X-shaped crystals under N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (B-DPPE) on 10 mM HEPES, 250 mM NaCl, 10mM EDTA, pH 7.8. (b) Wild-type streptavidin under B-DPPE in Nanopure water. Gray scale analysis demonstrates that the protein binds to the interface at similar levels but does not crystallize. (c) K132L forms rectangular crystals under B-DPPE on 10 mM HEPES, 250 mM NaCl, 10 mM EDTA, pH 7.8. (d) K132L forms rectangular crystals on Nanopure water. to remove buffer and counterions. Crystals appeared 5-10 min after protein injection. Brewster Angle Microscopy. Monolayer binding and crystal formation was monitored via a home-built Brewster angle microscope, for which the mechanical setup was based on a previously described design.23 The laser and polarizer are linearly arranged along a single axis, eliminating the need for additional mirrors or prisms. A 1 mm diameter beam of light from a 10 mW He-Ne laser was p-polarized using a dichroic sheet polarizer (extinction ratio ) 10-4) and then reflected from the air/water interface at an angle of incidence of 53.12° with respect to the surface normal, i.e., the Brewster angle for the air/water interface. A portion of the reflected light was focused directly on the sensing unit of a CCD camera (CCD-72 with active area 6.6 × 8.8 mm, Dage-MTI, Michigan City, IN). A custom configuration for the focusing optics provided a combination of long working distance (ca. 50 mm) to avoid interference with the barriers of the Langmuir trough, a relatively high magnification (ca. 26×) to visualize crystals typically 20-200 µm in size, and a largely (23) Cohen-Stuart, M. A.; Wegh, R. A.; Kroon, J. M.; Sudho¨lter, E. J. R. Langmuir 1996, 12, 2863-2865.

uniform and speckle-free image illumination to allow reliable, quantitative gray scale analysis. With this setup, the lateral resolution is approximately 5 µm. The images from the CCD camera were captured at 18 Hz with a SG-9 Scion video capture card (Scion Corp. Frederic, MD) in a StarMax 3000/180 PowerPC personal computer running NIH-image 1.60. Reflected intensity in gray scale units was continuously measured from unprocessed images before and after crystals appeared. The gray scales of the noncrystalline and crystalline phases were measured separately, as previously described.12 Using a previously developed model to quantitatively interpret BAM gray scales of streptavidin binding and crystallization beneath a biotinylated lipid monolayer,12 changes in gray scale were converted first into changes in refractive index of the protein layer and then into changes in the protein surface density. Thus changes in protein surface density with time could be calculated for both noncrystalline and crystalline phases.12 Image background subtraction was not performed. Transmission Electron Microscopy. The LangmuirScha¨fer 24 method was used to transfer crystals to 400 mesh copper grids covered with an ultrathin carbon film (Ted Pella, Redding, CA) for transmission electron microscopy (TEM). The

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Figure 3. Real image analysis of K132L crystals. (a) TEM image of crystals grown on 10 mM HEPES, 250 mM NaCl, 10 mM EDTA, pH 7.8. (b) Fourier transform of image (a), where reciprocal lattice vectors a* and b* for the C222 lattice were assigned by comparing the intensities of the observed diffraction pattern and those calculated from a molecular model of the C222 structure. (c) TEM image of crystals grown on Nanopure water. (d) Fourier transform of image (c), with a* and b* as indicated. grids were rinsed in chloroform to remove the Formvar backing immediately before placing them on the surface of the trough. The samples were then stained with 2% uranyl acetate for 1 min and imaged with a JEOL 1200 transmission electron microscope. Images were digitized using an Argus II flatbed scanner and Adobe Photoshop software at 1200 ppi resolution. 1024 × 1024 pixel sections of the digitized images, which correspond to 394 × 394 nm of crystal, were Fourier transformed with NIH Image 1.55 VDM software to determine unit cell parameters.

Results and Discussion The wild-type streptavidin formed the familiar X-shaped crystal as expected in the buffer system at both pH 7.8 and 6.5. The K132L mutant also formed crystals in the buffer system at both pH values, but they were rectangular in morphology. Unlike wild-type streptavidin, K132L also formed elongated rectangular crystals in the pure water subphase. While no crystals were ever observed with the wild-type streptavidin, the protein bound to the biotinylated lipid monolayer at densities >75% of the density of wild-type crystals observed in the buffered subphase (Figure 2). Gray scale analysis of BAM images revealed that wildtype protein in the buffer subphase rapidly adsorbs to the lipid layer and then crystallizes in 2D when the noncrystalline region reaches a critical surface density of 75% of (24) Langmuir, I.; Schaefer, V. J. J. Am. Chem. Soc. 1938, 59, 28032810.

the protein density in the crystal. This critical surface density value is independent of protein bulk concentration.12 K132L also crystallizes at a relative protein surface density of 75%, regardless of subphase ionic strength or the bulk protein concentration. Over a concentration range of 19-77 nM, the gray scale of both the crystalline and the noncrystalline phases remained constant with time after the crystals first appeared and grew to cover the surface, supporting previous observations with streptavidin that crystallization is a first-order phase transition.12 Crystallization results were the same for biotinylated lipids with a short linker, B-DPPE, and long linker, B-xDPPE. K132L crystals from the air-water interface were transferred to carbon grids for transmission electron microscopic analysis. Micrographs of transferred crystals were Fourier transformed to give the primitive lattice, which was then converted to a centered lattice. The resulting dimensions of the unit cell were a ) 84 ( 1 Å, b ) 83 ( 1 Å, and γ ) 90 ( 1° for core streptavidin crystals from the high ionic strength buffer at pH 7.8; a ) 84 ( 1 Å, b ) 84 ( 1 Å, and γ ) 90 ( 1° for the K132L crystals from the same buffer at pH 7.8; and a ) 87 ( 1 Å, b ) 84 ( 1 Å, and γ ) 90 ( 2° for the K132L crystals from water (Figure 3). These spacings match the previously described C222 crystal form obtained with commercial streptavidin preparations.1,3 Thus we expect the protein-protein

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contacts in the K132L crystals (with or without salt) are very similar to those in the native X-shaped crystals. These results have important mechanistic implications for the 2D streptavidin crystallization process. The remarkable ability of the K132L mutant to crystallize from a water subphase demonstrates that this single unfavorable electrostatic interaction between the Lys 132 amine groups is a barrier to crystallization and underlies the counterion dependency of streptavidin crystallization. Chloride counterions have been present in all previous reports of streptavidin two-dimensional crystallization under biotinylated or charged lipid monolayers and likely serve as the necessary counterions in these experiments. We also conducted studies in the HEPES buffer without NaCl and found that wild-type and K132L streptavidin again form X-shaped and rectangular crystals, respectively. This suggests that the HEPES anion can also serve as the counterions, and our molecular modeling indicates that the sulfonic acid moiety can readily fit into the interface to shield the lysine 132 amine groups. It thus appears that a variety of anionic species might serve as appropriate counterions. These results also suggest that a kinetic barrier to protein-protein association is a key determinant of macroscopic crystal morphology, as the alteration of Lys 132 results in a change from X-shaped to rectangular morphology while the C222 lattice symmetry, and thus a similar set of protein-protein interfaces, are retained. It has been hypothesized that the X-shaped morphology could be the result of dendritic crystal growth caused by the presence of noncrystallizing protein components found in commercially prepared streptavidin.25 Our finding that the recombinant core streptavidin (residues 13-139) also yields the familiar X-shaped morphology suggests that a different mechanism must be responsible, as the recombinant protein is a single molecular weight species to the resolution of 2-3 amu by high-resolution electrospray mass spectrometry. The formation of counterion-Lys 132 interactions and the remainder of the crystal contact minimally includes the following events: the diffusional (25) Ku, A. C.; Darst, S. A.; Kornberg, R. D.; Robertson, C. R.; Gast, A. P. Langmuir 1992, 8, 2357-2360. (26) Boodhoo, A.; Duke, N. E. C.; Kong, D.; Ritzel, M. W. L.; Kunimoto, D. Y.; Read, R. J. J. Mol. Biol. 1994, 241, 269-272. (27) Lawson, D. M.; Artymiuk, P. J.; Yewdall, S. J.; Smith, J. M. A.; Livingstone, J. C.; Treffry, A.; Luzzago, A.; Levi, S.; Arolsio, P.; Cesareni, G.; Thomas, C. D.; Shaw, W. V.; Harrison, P. M. Nature 1991, 349, 541-544. (28) Braig, K.; Otwinowski, Z.; Hedge, R.; Boisvert, D. C.; Joachimiak, A.; Horwich, A. L.; Sigler, P. B. Nature 1994, 371, 578-586. (29) Vijay-Kumar, S.; Sendahi, S. E.; Ealick, S. E.; Nagabhushan, T. L.; Trotta, P. P.; Kosecki, R.; Reichert, P.; Bugg, C. E. J. Biol. Chem. 1987, 262, 4804-4805.

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encounter of proteins and counterions, the desolvation of anions and closely interacting protein surfaces, and the formation of noncovalent bonds. Removing the need for formation of the counterion-protein interaction results in a kinetic barrier alteration at a key intermediate(s) along this crystallization reaction coordinate that is involved in determining the crystal morphology. The precise kinetic intermediate(s) cannot be identified at this point, but the change in crystal morphology could for example occur through changes of the rate of addition of unincorporated protein to the existing crystal. Alternatively, the key intermediate could be involved in early nucleation events where the counterion-Lys 132 interaction could control the rate of association of protein clusters, e.g., an early twinning nucleus could be altered by the different rate of protein-protein association when the counterion requirement is removed. Conclusions Re-engineering three-dimensional protein contacts has previously enabled crystallization of difficult to crystallize proteins or yielded new crystals with improved diffraction properties.26-29 Our results provide experimental evidence that 2D streptavidin crystal morphology is reaction controlled and that formation of the counterion-mediated Lys 132 electrostatic interaction is a key determinant of the energetic barrier(s) controlling crystal shape. While the direct study of protein-protein molecular recognition in 3D crystallization is made difficult by crystal polymorph complexity, we have shown here that the orientation specificity induced by interfacial binding in the twodimensional system facilitates the characterization of specific side-chain roles at the protein-protein contact interface. Continued study of this 2D system may thus provide a better understanding of how specific proteinprotein interactions and protein-protein association/ dissociation kinetics are related to crystal nucleation and growth kinetics. This should in turn provide useful insight for the development of better crystallization strategies and in addition may provide new avenues for controlling protein assembly processes at interfaces in diagnostic, drug delivery, and biomaterial applications. Acknowledgment. We gratefully acknowledge the support of NASA (NAG8-1149), the National Science Foundation (through the UWEB Engineering Research Center), and the Pharmaceutical Research and Manufacturers Association of America (postdoctoral fellowship to S.K.). LA9801918