Molecular Self-Assembly of Solid-Supported Protein Crystals

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Molecular Self-Assembly of Solid-Supported Protein Crystals Chengfei Lou, Matthew Shindel, Landon Graham, and Szu-Wen Wang* Department of Chemical Engineering and Materials Science, UniVersity of California, IrVine, California 92697-2575 ReceiVed February 5, 2008. ReVised Manuscript ReceiVed May 5, 2008 Highly ordered protein arrays have been proposed as a means for templating the organization of nanomaterials. Toward this end, we investigate the ability of the protein streptavidin to self-assemble into various configurations on solid-supported phospholipids. We identify two genetic variants of streptavidin (comprising amino acids 14-136 and 13-139) and examine their molecular organization at the liquid-solid interface. Our results demonstrate that the structural differences between these two protein variants affect both crystalline lattice and domain morphology. In general, these results for the liquid-solid interface are similar and consistent with those at the air-water interface with a few notable differences. Analogous to crystallization at the air-water interface, both forms of streptavidin yield H-like domains with lattice parameters that have C222 symmetry at pH 7. At pH 4, the native, truncated form of streptavidin yields needle-like domains consisting of molecules arranged in P1 symmetry. Unlike crystalline domains grown at the air-water interface, however, the lattice parameters of this P1 crystal are unique and have not yet been reported. The presence of a solid substrate does not appear to dramatically alter streptavidin’s two-dimensional crystallization behavior, suggesting that local intermolecular interactions between proteins are more significant than interactions between the interface and protein. Our results also demonstrate that screening the electrostatic repulsion between protein molecules by modulating ionic strength will increase growth rate while decreasing crystalline domain size and macroscopic defects. Finally, we show that these domains are indeed functional by attaching biotinylated gold nanoparticles to the crystals. The ability to modulate molecular configuration, crystalline defects, and domain size on a functional array supports the potential application of this system toward materials assembly.

Introduction Control of physical arrangement and distances between inorganic nanoparticles offers a new avenue for fabricating materials with novel properties. At interparticle spacings below 10 nm, electron tunneling can affect energy transfer between nanocrystals and give rise to unique optical, electronic, and magnetic properties.1 These special characteristics have potential in future generations of memory and optical devices,2–4 electrochemical systems,5 and catalytic applications.6,7 An existing challenge, however, is the ability to manipulate these particles into a defined spatial arrangement at the molecular level. One approach for addressing this difficulty has been to exploit natural self-associating biological systems to template arrays of inorganic nanoparticles with a defined architecture. These natural structures are precise and correspond to the nanometer length scales needed for organization. Recently, protein systems using chaperonins8 and S-layers9,10 have been manipulated to pattern prefabricated nanoparticles into highly ordered arrays. These * Corresponding author. Phone: 949-824-2383. Fax: 949-824-2541. E-mail: [email protected]. (1) Murray, C. B.; Kagan, C. R.; Bawendi, M. G. Annu. ReV. Mater. Sci. 2000, 30, 545–610. (2) Zhirnov, V. V.; Herr, D. J. C. Computer 2001, 34, 34–43. (3) Xia, Y.; Gates, B.; Yin, Y.; Lu, Y. AdV. Mater. 2000, 12, 693–713. (4) Eychmuller, A. Organization of Nanoparticles. In Nanoparticles: From Theory to Application; Schmidt, G., Ed.; Wiley-VCH Verlag GmbH & Co.: New York, 2004. (5) Tuller, H. L. J. Electroceram. 1997, 1(3), 211–218. (6) Grunes, J.; Zhu, J.; Yang, M.; Somorjai, G. A. Catal. Lett. 2003, 86(4), 157–161. (7) Grunes, J.; Zhu, J.; Anderson, E. A.; Somorjai, G. A. J. Phys. Chem. B 2002, 106, 11463–11468. (8) McMillan, R. A.; Paavola, C. D.; Howard, J.; Chan, S. L.; Zaluzec, N. J.; Trent, J. D. Nat. Mater. 2002, 1, 247–252. (9) Hall, S. R.; Shenton, W.; Engelhardt, H.; Mann, S. Chemphyschem 2001, 2(3), 184–186. (10) Mark, S. S.; Bergkvist, M.; Yang, X.; Teixeira, L. M.; Bhatnagar, P.; Angert, E. R.; Batt, C. A. Langmuir 2006, 22(8), 3763–3774.

types of proteins have also served as nucleation sites which enable particles to be directly grown into organized architectures.11,12 Alteration of spatial orientation in these investigations was performed by changing the microbial source from which the protein originates. We investigate an alternative protein system which has been shown to yield different two-dimensional lattices under various conditions. Streptavidin, with its unusually high binding affinity for the ligand biotin and its tetrameric subunits arranged in 222 point group symmetry,13 makes it a candidate for enabling higherorder nanostructure assembly. A number of studies investigating streptavidin assembly at the air-water interface have been reported, and it has been found that the molecular configuration of these arrays can be manipulated by both environmental factors14–16 and genetic engineering.17–20 Low pH values yield monolayer protein crystals with compact packing (P1 symmetry) and needle-like morphologies, while crystals grown at high pH result in a square lattice arrangement (C222 symmetry) with Xor H-shaped morphologies. Intermediate pH values result in crystals with transitional morphologies exhibiting P2 lattice (11) Shenton, W.; Pum, D.; Sleytr, U. B.; Mann, S. Nature 1997, 389, 585– 587. (12) Sleytr, U. B.; Egelseer, E. M.; Ilk, N.; Pum, D.; Schuster, B. FEBS J. 2007, 274(2), 323–334. (13) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85–88. (14) Wang, S. W.; Poglitsch, C. L.; Yatcilla, M. T.; Robertson, C. R.; Gast, A. P. Langmuir 1997, 13(22), 5794–5798. (15) Gast, A. P.; Robertson, C. R.; Wang, S. W.; Yatcilla, M. T. Biomol. Eng. 1999, 16(1-4), 21–27. (16) Ratanabanangkoon, P.; Gast, A. P. Langmuir 2003, 19, 1794–1801. (17) Wang, S. W.; Robertson, C. R.; Gast, A. P. Langmuir 1999, 15(4), 1541– 1548. (18) Wang, S. W.; Robertson, C. R.; Gast, A. P. J. Phys. Chem. B 1999, 103(37), 7751–7761. (19) Wang, S. W.; Robertson, C.; Gast, A.; Koppenol, S.; Edwards, T.; Vogel, V.; Stayton, P. Langmuir 2000, 16(11), 5199–5204. (20) Farah, S. J.; Wang, S. W.; Chang, W. H.; Robertson, C. R.; Gast, A. P. Langmuir 2001, 17(19), 5731–5735.

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symmetry or interspersed cocrystallization (P1, P2, C222) within a single-crystalline domain. Molecular configuration can also be manipulated by modifying specific intermolecular interactions, at the N- and C-termini or within the protein, through protein engineering. Since the molecular configuration of our existing crystalline protein system can be altered, we envision the ability to define long-range ordering of inorganic particles by manipulating this protein template. To do this, we need to grow relatively large, functional two-dimensional protein monolayers with minimal molecular defects and understand how size, quality, and molecular configuration of crystals can be controlled. While crystallization at the air-water interface is useful for probing the fundamentals of protein array fabrication, practical applications require crystalline monolayers to be grown on solid substrates. Whether the two-dimensional crystallization behavior of streptavidin at the air-water interface is translatable to solidsupported organization, however, is not obvious a priori. Previous investigations with S-layer proteins which crystallize on solid substrates and at the air-water interface have shown that the properties of the surfaces onto which the proteins bind and selfassemble can affect crystallization and molecular-level orientation. Key attributes of these surfaces include their charge21 and hydrophobicity.22,23 Furthermore, the amphiphilic nature of proteins often causes them to denature at surfaces, including the air-water24,25 and liquid-solid26,27 interfaces. Since we are organizing our protein molecules at these different surfaces, it is possible that protein conformational changes could be generated by its interaction with the surface, which in turn could affect crystallization. Finally, the diffusivity at the air-water interface is approximately an order of magnitude higher (∼10-7 cm2/ s)28,29 than at the liquid-solid interface (∼10-8 cm2/s).30–32 Since variations in mass-transport properties can alter crystal morphologies33 (in particular of two-dimensional streptavidin crystals34), it is possible that such diffusivity differences could also influence morphological development and self-assembly behavior. In this work, we examine the extent to which the presence of a solid substrate can affect the self-assembly of streptavidin into defined arrays. Our focus is to determine the effects of interaction changes between proteins bound at the liquid-solid interface and examine the resulting molecular configuration and crystal quality relative to events at the air-liquid interface. Interactions are manipulated by changing the pH and ionic strength of the liquid phase and using genetic variants that differ in their protein-protein contacts within arrays. Furthermore, we seek to determine whether the proteins which comprise these solid(21) Wetzer, B.; Pfandler, A.; Gyorvary, E.; Pum, D.; Losche, M.; Sleytr, U. B. Langmuir 1998, 14(24), 6899–6906. (22) Pum, D.; Sleytr, U. B. Supramol. Sci. 1995, 2(3-4), n/a. (23) Pum, D.; Weinhandl, M.; Hodl, C.; Sleytr, U. B. J. Bacteriol. 1993, 175(9), 2762–2766. (24) Postel, C.; Abillon, O.; Desbat, B. J. Colloid Interface Sci. 2003, 266(1), 74–81. (25) Adams, S.; Higgins, A. M.; Jones, R. A. L. Langmuir 2002, 18(12), 4854–4861. (26) Sethuraman, A.; Han, M.; Kane, R. S.; Belfort, G. Langmuir 2004, 20(18), 7779–7788. (27) Czeslik, C.; Winter, R. Phys. Chem. Chem. Phys. 2001, 3(2), 235–239. (28) Bohorquez, M.; Patterson, L. K. J. Phys. Chem. 1988, 92(7), 1835–1839. (29) Kim, S. H.; Yu, H. J. Phys. Chem. 1992, 96(10), 4034–4040. (30) Subramaniam, S.; Seul, M.; Mcconnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83(5), 1169–1173. (31) Calvert, T. L.; Leckband, D. Langmuir 1997, 13(25), 6737–6745. (32) Lou, C.; Wang, Z.; Wang, S.-W. Langmuir 2007, 23, 9752–9759. (33) Davey, R. J.; Garside, J. From Molecules to Crystallizers; Oxford University Press: New York, 1999. (34) Ku, A. C.; Darst, S. A.; Kornberg, R. D.; Robertson, C. R.; Gast, A. P. Langmuir 1992, 8(10), 2357–2360.

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supported crystalline domains are functional by binding biotinylated nanoparticles to them.

Materials and Methods Materials. Phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (DTPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were obtained from Avanti Polar Lipids, and N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoehtanolamine (biotin-X-DHPE) was purchased from Invitrogen. Fluorescein5-isothiocyanate (FITC), Sephadex G50, and glutaraldehyde were from Sigma-Aldrich. Buffer reagents (NaH2PO4, NaCl, NaNO3) and solvents (chloroform, methanol, dimethylsulfoxide) were purchased from EMD Chemicals, and HCl and NaOH were from Fisher. Both hydrogen peroxide (30% w/w) and concentrated sulfuric acid were also purchased from Fisher. To functionalize gold nanoparticles with biotin, we started with colloidal gold (monodispersed, 20 nm average particle size) from Ted Pella. 11-Mercaptoundecylamine (11-MUAM) was obtained from Dojindo. N-Hydroxy-succinimide-poly(ethylene oxide)4-biotin (NPB) was provided by Pierce. 11-Mercaptoundecanoic acid (11MUDA), 200 proof ethanol, and triethanolamine (TEA) were from Aldrich, and thioctic acid (TA) was from Sigma. N,N-Dimethylformamide (DMF) was acquired from Omnisolve. All glassware was cleaned with a ‘piranha solution’ (1:3 30% hydrogen peroxide/ concentrated sulfuric acid) before use. (CAUTION: Piranha solution reacts Violently with most organic materials and should be handled with extreme care.) Water was purified with a Milli-Q purification system with a final specific resistivity > 18 MΩ/cm (Millipore). For atomic force microscopy (AFM), we used grade V1 mica (Ted Pella), HI’RES AFM tips (MikroMasch) for high-resolution imaging, and STING tips (MikroMasch) for imaging nanoparticles. Two different forms of streptavidin were used. Recombinant streptavidin (SA-R) produced from E. coli was from Roche Applied Science and spans amino acids 13-139. Native, core streptavidin (SA-N) from the natural bacterial source Streptomyces aVidinii was a gift from Prozyme. According to Prozyme, this source yields a truncated form comprising primarily amino acids 14-136 with a minor form of residues 14-137. Mass spectrometry on both proteins to confirm molecular weight and protein sequence was performed on a MALDI-TOF mass spectrometer (Perseptive Biosystems) after samples were dialyzed in deionized water. Streptavidin Crystallization at the Air-Water Interface. For streptavidin crystals grown directly at the air-water interface, we followed previously reported procedures.14,34 Buffer containing 500 mM NaCl and 50 mM NaH2PO4, with the pH adjusted to 4 or 7 with HCl or NaOH, was filtered with a 0.2 µm filter prior to filling a miniature, 1.4-mL Langmuir trough. A lipid mixture dissolved in chloroform and consisting of 5 mol % biotin-X-DHPE in 95% DOPC was spread on the aqueous surface at an estimated surface pressure of ∼25-30 mN/m.14 Streptavidin (either SA-N or SA-R) was labeled with FITC to an average ratio of 1.2-1.5 molecules of FITC per molecule of tetrameric protein.32,35 The protein was injected beneath the phospholipid monolayer to a final concentration of 15-20 µg/mL, which was the minimum concentration that consistently yielded crystalline domains. Crystal growth was observed with fluorescence microscopy (BX51, Olympus), and images were recorded with a cooled CCD camera (ORCA-285, Hamamatsu) after 4 h of incubation. Streptavidin Crystallization at the Liquid-Solid Interface. The protocol for solid substrate functionalization and streptavidin crystallization has been previously described.31,32 In summary, mica surfaces were functionalized using Langmuir-Blodgett deposition, in which the first leaflet was DPPE and the second leaflet consisted of 5 mol % biotin-X-DHPE diluted in 95 mol % DTPC. This biotinX-DHPE concentration yielded confluent crystal coverage, and lower lipid ratios of 3% biotin-X-DHPE and 97% DTPC were used to (35) Nargessi, R. D.; Smith, D. S. Methods Enzymol. 1986, 122, 67–72.

Molecular Self-Assembly of Solid-Supported Protein Crystals obtain individual crystal domains for morphological identification. The lipid-functionalized surface was incubated in a solution of 50 mM NaNO3 with 5 mM NaH2PO4, and the pH of the solution adjusted to 4 or 7 with addition of either HNO3 or NaOH. Streptavidin was added to obtain a final concentration of 7-8 µg/mL. After 4 h of incubation, crystals were imaged by fluorescence microscopy. To investigate the effect of ionic strength, the pH of the buffer was kept constant at 7 with 5 mM NaH2PO4 while the salt concentration was varied between 5 and 500 mM NaNO3. The final protein concentration was also kept constant at 7-8 µg/mL. Our data indicate that there are no apparent differences in the crystals formed using NaCl or NaNO3 salt at these conditions (Lou, C. Unpublished results). To be consistent with prior investigations,31,32 therefore, solid-supported crystal growth was performed with NaNO3. Crystals were grown to confluency using a 5% biotin-X-DHPE substrate and treated as described above. To characterize the molecular configuration of the two-dimensional crystals, glutaraldehyde was added to the buffer to a final concentration of 0.5% v/v and incubated overnight. After drying, samples were analyzed with atomic force microscopy (AFM, MultiMode SPM, Digital Instruments) in tapping mode. AFM images were processed with Nanoscope v 5.2 (Veeco) and Scion Image software as previously described.32 Fluorescence Recovery after Photobleaching (FRAP). To investigate the role of protein diffusion on crystallization kinetics at the different ionic strengths, we used fluorescence recovery after photobleaching (FRAP) to determine diffusion coefficients. Details of our experimental protocol have been described in a previous publication32 and are briefly summarized here. Since growth of crystals to a confluent monolayer precludes diffusion measurements, we performed FRAP at the noncrystallizing condition of 1% biotinX-DHPE in DTPC. Samples of streptavidin on mica-supported bilayers at different ionic strengths were prepared as described above. These samples were then transferred to buffer free of streptavidin to minimize fluorescent background. Regions were observed with a multispectral laser-scanning microscope (LSM 510 META, Zeiss) at room temperature (∼22 °C) and photobleached with a Ti:sapphire two-photon femtosecond laser (Mai Tai, Spectra-Physics) at a wavelength of 800 nm. Fluorescence intensity and recovery were recorded by LSM 510 software (Zeiss), and the data was processed and fit with Origin (OriginLab) according to the mathematical treatment of Axelrod.36 Surface Modification of Colloidal Gold Nanoparticles with Biotin. To biotinylate gold nanoparticles (AuNPs), we first synthesized biotin-poly(ethylene oxide)4-1-undecane-thiol (BPT) following a method published by Searson and co-workers.37 A 2 mM solution of 11-MUAM was incubated overnight with a 2-fold molar excess of NPB in a 1:1:0.04 (v/v) DMF/DMSO/TEA solvent mixture under an argon blanket. The BPT product solution was then transferred to a clean glass vial and stored in the dark at 4 °C. We then modified the surface chemistry of the gold nanoparticles using a protocol developed by Lin et al.38 In brief, 20 nm gold sols with an initial concentration of ∼1.2 nM were centrifuged and resuspended in water (pH was adjusted to 11 by NaOH and degassed with N2). TA (5 mM) in degassed ethanol was then added to the particle suspension in a volummetric ratio of 100:1 colloidal sol to TA. This reaction was carried out in the dark at room temperature under constant agitation for at least 18 h. The particles were washed to remove unreacted TA molecules by repeated cycles of centrifugation and resuspension in water (pH 11, to a concentration of 1.2 nM). The gold nanoparticles were then mixed with either a 5 mM solution of 30% (mol/mol) BPT and 70% (mol/mol) 11-MUDA in DMF:DMSO:EtOH (37.5%:37.5%:25%) (for biotinylated particles) or a 5 mM ethanolic solution of 11-MUDA (for control particles). The AuNP sol and thiol solutions were mixed in a 100:1 (v/v) ratio, (36) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E. L.; Webb, W. W. Biophys. J. 1976, 16(2), A217–A217. (37) Salem, A. K.; Chen, M.; Hayden, J.; Leong, K. W.; Searson, P. C. Nano Lett. 2004, 4(6), 1163–1165. (38) Lin, S. Y.; Tsai, Y. T.; Chen, C. C.; Lin, C. M.; Chen, C. H. J. Phys. Chem. B 2004, 108(7), 2134–2139.

Langmuir, Vol. 24, No. 15, 2008 8113 respectively. The subsequent ligand-exchange reaction was then agitated for a minimum of 18 h in the dark, and particles were subsequently washed by multiple cycles of centrifugation and resuspension. The particles were finally resuspended in a 5 mM phosphate buffer at pH 7 at a concentration of ∼12 nM. Attachment of Gold Nanoparticles to Protein Crystal. After crystallizing streptavidin on a mica-supported, phospholipid bilayer, the crystallization buffer was exchanged for a buffer of lower ionic strength (6 mM NaNO3, 5 mM phosphate, pH 7). Several washes were performed to ensure removal of free protein. Colloidal sol (biotinylated gold particles or nonbiotinylated control particles) were then added to the crystallization vessel, which contained the solidsupported protein crystals, to a final concentration of 1 nM. The solution was then gently pipetted for several minutes to thoroughly mix the particles, followed by 2.5 h of incubation. The samples were washed with fresh buffer and cross-linked overnight with glutaraldehyde (0.5%). After rinsing with water and drying, samples were imaged with AFM as described above.

Results and Discussion a. Variants of Core Streptavidin. Streptavidin produced natively by the bacteria Streptomyces aVidinii has been reported to undergo post-translational modifications. These variants of different lengths, referred to as “core” streptavidin, result from cleavage at amino acids 13 or 14 at the N-terminus and between residues 133 and 139 at the C-terminus.13,39 Tailored variants of streptavidin can also be created by genetic engineering, which enables us to specifically define internal sequences for mutagenesis as well. Previously reported two-dimensional crystallization investigations performed at the air-water interface show that these Nand C-termini (as well as other specific internal amino acid sites) are important protein-protein contacts in monolayer selfassembly.18,19 By designing different molecular forms of streptavidin that are truncated at various terminal sites, the order and crystal lattice of the array can be modulated, giving crystals with P1, P2, or C222 symmetry. These prior studies were performed at the air-water interface, and we desired to examine the extent to which introduction of a solid substrate affects the modulation of streptavidin selfassembly. In this study, we focus on two different molecular forms of streptavidin that were previously determined to generate different crystalline lattices. Experiments are performed with recombinant streptavidin 13-139 (produced in E. coli) and commercial streptavidin (predominately amino acids 14-136 from the native S. aVidinii). In light of the crystallization differences due to protein forms, it is important to use the same form when comparing crystallization data across different substrate experiments. This may not be straightforward, as commercial streptavidin sold by different vendors often does not come with complete information regarding post-translational processing and the identity of the final form. Since the original sources of streptavidin for prior air-water experiments were no longer available, we examined commercial sources of streptavidin and obtained two forms which likely corresponded to the variants described in prior studies. Core streptavidin obtained from Roche (SA-R) is a recombinant form that should span amino acids 13-139, while native streptavidin from Prozyme (SA-N) is described by the vendor to be a combination of variants 14-136 (major component) and 14-137 (minor component). We used mass spectrometry to confirm these protein lengths. The mass of SA-R was measured to be 13 273 ( 1.4 Da, which (39) Pahler, A.; Hendrickson, W. A.; Kolks, M. A.; Argarana, C. E.; Cantor, C. R. J. Biol. Chem. 1987, 262(29), 13933–13937.

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Table 1. Summary of Streptavidin Monolayer Crystals at the Air/Water Interface previously reported crystal forms at air-water form

streptavidin variant (amino acids)

SA-N

native “core” from S. aVidinii (14-136)

SA-R

recombinant from E. coli (13-139)

pH 4 7 4 7

is consistent with the predicted mass of 13 271 Da and corresponds to a protein which spans amino acids 13-139. For SA-N, the major peak yielded a mass of 12 974 ( 2.4 Da and a minor peak gave 13 044 ( 2.8 Da; these are close to the predicted molecular weights of 12 971 and 13 042 for streptavidin forms 14-136 and 14-137, respectively. These values indicate, therefore, that these two streptavidin variants are comparable to those used in prior air-water experiments. Our investigation demonstrates that divergent self-assembling behavior, including differences in crystalline lattices and morphologies, can be obtained which depends solely on the protein variant. Therefore, given the large number of studies in the literature of streptavidin bound to surfaces, it may be important to determine the form of protein used and consistently obtain protein from the same commercial source. b. Effects of pH and Protein Length on Assembly at the Air-Water Interface. Prior experiments at the air-water interface using streptavidin 13-139 and streptavidin 14-136 showed that both the C- and N-terminal ends of streptavidin are at protein-protein interfaces in all three major crystal types (P1, P2, and C222).19 Such subtle changes to these ends can yield a significant effect on molecular packing, and a summary of these previous results is presented in Table 1. While the presence of H/X-shaped C222 crystals are observed at pH 7 for both forms, disparity between protein-protein interactions at pH 4 yields high-aspect, needle-like P1 crystal for streptavidin 14-136 and H/X-shaped P2 crystal for streptavidin 13-139. These crystal morphologies were found to correlate to the molecular lattice such that the lattice parameters could be surmised from observation of macroscopic domain shape.17–19 As a baseline comparison and to further confirm that the two forms of streptavidin we have recently obtained (SA-R and SA-N, from sources different from prior experiments) do indeed exhibit previously reported crystallization behavior, we tested these proteins at the air-water interface. The results from our two current forms of protein match published data, and they are presented in Figure 1. Since the morphology of these crystals is correlated to molecular configuration, we used domain shape to confirm crystallization behavior. As expected, the native, truncated form of core streptavidin (SA-N) yielded needle-like structures at pH 4 and H-shaped and rectangular domains at pH 7. Streptavidin with three extra amino acids at the C terminus (SA-R) also resulted in X- and H-shaped domains at pH 7 but at pH 4 yielded H-shaped domains. Given the mass spectrometry and air-water crystallization data, therefore, we conclude that our current commercial sources of streptavidin are comparable with the proteins previously used and therefore allow us to extend our studies to assembly at the liquid-solid interface. c. Effects of pH and Protein Length on Assembly at the Liquid-Solid Interface. We examined the effect of a solid substrate on crystal monolayer growth at the same conditions as the previous experiments at the air-water interface. In general, the results for the liquid-solid interface are similar and consistent with those at the air-water interface with some minor differences. Details are described in the sections below and summarized in Table 2.

crystal morphology needles, high aspect ratios H, X, or rectangular H- or X-shaped H- or X-shaped

unit cell parameters

references

P1 C222 P2 P2 and C222

19, 56 19, 57 19 19

Figure 1. Macroscopic domains of streptavidin grown at the air/water interface. As expected, the two different forms yielded different crystal morphologies at pH 4 and 7. At pH 4, (A) native, truncated streptavidin (SA-N, spanning residues 14-136) yields needle-like crystals while (B) the recombinant form (SA-R, spanning residues 13-139) yields H-shaped crystals. At pH 7, (C) SA-N yields H-shaped or rectangular crystals and (D) SA-R gives H-shaped and X-shaped domains. The optical anisotropy observed for these domains under a polarizer analyzer confirms the molecular order of the monolayer within the crystalline domain. Scale bar is 50 µm.

NatiVe, Truncated Form of StreptaVidin (SA-N, 14-136) at pH 4 Yields Needle-like Domains with Altered P1 Symmetry. At the air-water interface, core streptavidin, corresponding to amino acids 14-136, has been shown to yield needle-like domains with defined P1 symmetry corresponding to unit cell parameters of a ) 5.8 nm, b ) 5.0 nm, and γ ) 113°.17,40 Under the same conditions of crystal growth on a solid substrate, this truncated form of streptavidin also gives similar macroscopic morphologies. As shown in Figure 2, we observe large needle-like domains surrounded by numerous smaller domains of similar aspect ratios. At the molecular level, these proteins are also packed into an arrangement with P1 symmetry; however, the crystalline lattice is expanded with parameters of a ) 6.9 ( 1.1 nm, b ) 5.7 ( 0.8 nm, and γ ) 101.9 ( 5.3°. This configuration has not yet been reported in any streptavidin monolayer studies. Statistical analysis comparing the lattice cell parameters of this crystal with P1 symmetry with those of the previously reported P1 crystal32 (Table 2) indicate that they are different within a 94% confidence interval. NatiVe, Truncated Form of StreptaVidin (SA-N, 14-136) at pH 7 Yields H-like Domains with C222 Symmetry. Our data show that truncated streptavidin behaves similarly in pH 7 at both the air-water and liquid-solid interfaces (Figure 3). Under both conditions, H-shaped morphologies form, resulting from molecules packed in the symmetric, C222 square-lattice arrangement. Unit cell parameters for the solid-supported crystals were a ) 6.3 ( 0.1 nm, b ) 6.2 ( 0.1 nm, and γ ) 91.8 ( 0.9°. These values are consistent with the parameters previously reported for crystal growth directly on a solid mica support (for (40) 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(2), 308–316.

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Table 2. Summary of Streptavidin Monolayer Crystals at the Liquid/Solid Interface biotin conc.

salt conc. (mM NaNO3)

pH

streptavidin form

crystal

5% 5% 5% 5% 5% 5% 1.5%

50 50 50 50 5 500 50

4 7 4 7 7 7 7

SA-N (14-136) SA-N (14-136) SA-R (13-139) SA-R (13-139) SA-R (13-139) SA-R (13-139) SA-R (13-139)

new P1 (a ) 6.9 ( 1.1 nm, b ) 5.7 ( 0.8 nm, γ ) 101.9 ( 5.3°) C222 crystalline, but no lattice parameters available C222 C222; large domains with macroscopic holes in the crystal C222; relatively small domains P1 (a ) 5.7 ( 0.1 nm, b ) 5.0 ( 0.2 nm, γ ) 110 ( 0.1°)a

a

The effect of biotin concentration was separately examined in a recent publication.32

Figure 2. Crystals grown on a solid substrate at pH 4 with streptavidin form SA-N (residues 14-136). (A) Macroscopic domains yield needle-like morphologies at 3% biotin-X-DHPE/97% DTPC. Scale bar is 50 µm. (B-D) AFM images of confluent crystals show a crystal form not previously reported with P1 symmetry. (B) Raw image of SA-N crystals at pH 4, (C) its enhanced, reconstructed image, and (D) the corresponding diffraction pattern. This new or altered P1 crystal has lattice parameters of a ) 6.9 ( 1.1 nm, b ) 5.7 ( 0.8 nm, γ ) 101.9 ( 5.3°. AFM scan size in B and C is 200 nm.

Figure 3. Crystals grown on a solid substrate at pH 7 with streptavidin form SA-N (residues 14-136). (A) Macroscopic domains yield dendritic-H morphologies at 3% biotin-X-DHPE/97% DTPC. Scale bar is 50 µm. (B-D) AFM images of confluent crystals show that crystals have C222 symmetry with expected lattice parameters. (B) Raw image of SA-N crystals at pH 7, (C) its enhanced, reconstructed image, and (D) the corresponding diffraction pattern. Lattice parameters are a ) 6.3 ( 0.1 nm, b ) 6.2 ( 0.1 nm, γ ) 91.8 ( 0.9°. AFM scan size is 200 nm.

one streptavidin tetramer per unit cell) and imaged with AFM.32 Furthermore, they are also very close to, although slightly larger than, unit cell parameters obtained for monolayers grown at the air-water interface (for one tetramer per unit cell).40 Recombinant Form of StreptaVidin (SA-R, 13-139) at pH 4 Yield H-like and Wide Needle-like Domains. At the air-water interface, SA-R at pH 4 yields H- or X-shaped morphologies that are formed from single P2 crystals. At the liquid-solid interface, we observe large dendritic crystals which are surrounded by smaller dendritic-H and wide needle-like domains (Figure 4). The presence of dendrites is attributed to transport-limited growth.34 All crystals grown under these conditions, however, were fragile and fractured easily, which resulted in crystalline domains that were not large enough to perform diffraction analysis and definitively identify crystal form. This fracturing most likely occurred during the transfer and drying process of AFM sample preparation. Experimental evidence supports these crystals to be either P2 or a coexistence of P1 and C222 crystals. AFM images of small local regions which remained intact (on the length scale of a few protein molecules) suggested that both P1 and C222 lattices were present; these domains, however, were too small to confirm unit cell parameters via diffraction analysis. In addition to crystal coexistence of P1 and C222, this observation could also support P2 crystals since the known P2 form has elements of both P1 and C222 lattice parameters.14,17 Furthermore, air-water experi-

Figure 4. Crystals grown on a solid substrate at pH 4 with streptavidin form SA-R (residues 13-139). Macroscopic domains yield occasional large dendritic crystals surrounded by numerous smaller, dendritic-H and wide needle-like domains. These crystals are very fragile, and AFM analysis of these domains did not yield regions that were large enough to perform diffraction analysis. Scale bar is 50 µm.

ments have shown that P2 crystals are very fragile relative to C222.14 Although other investigators have observed streptavidin crystals with P2 symmetry on mica,41 we have not been able to confirm the presence of this crystal form in our solid substrate studies. Differences between buffer composition or AFM sample (41) Reviakine, I.; Brisson, A. Langmuir 2001, 17(26), 8293–8299.

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Figure 5. Crystals grown on a solid substrate at pH 7 with streptavidin form SA-R (residues 13-139). (A) Macroscopic domains yield H-shaped morphologies at 3% biotin-X-DHPE/97% DTPC. Scale bar is 50 µm. (B-D) AFM images of confluent crystals show that crystals have C222 symmetry with expected lattice parameters. (B) Raw image of SA-R crystals at pH 7, (C) its enhanced, reconstructed image, and (D) the corresponding diffraction pattern. Lattice parameters are a ) 6.3 ( 0.2 nm, b ) 6.2 ( 0.2 nm, γ ) 90.4 ( 2.9°. AFM scan size is 200 nm.

preparation and data acquisition could account for this discrepancy. Recombinant Form of StreptaVidin (SA-R, 13-139) at pH 7 Yield H-like Domains with C222 Symmetry. Recombinant streptavidin (SA-R) at the air-water interface was reported to yield H- and X-morphologies at near-physiological pH. At pH 7, these domains comprised a combination of P2 and C222 crystals, while a pH above 7.8 they yielded solely the C222 arrangement.19 On a solid surface, we observed that all SA-R crystals at pH 7 exhibited C222 symmetry with lattice parameters of a ) 6.3 ( 0.2 nm, b ) 6.2 ( 0.2 nm, and γ ) 90.4 ( 2.9° (Figure 5). These unit cell values are consistent with those obtained for the native, truncated streptavidin (SA-N) at pH 7. At neutral pH, therefore, there appears to be little influence of the protein form or commercial source. Comparison of Results at the Air-Water and Liquid-Solid Interfaces. While there are indeed a few noted differences between crystal growth on an air-liquid surface compared to a liquid-solid surface, growth at these two interfaces for the most part are similar. The extent to which crystallization behavior is comparable at the different interfaces is not obvious a priori. As discussed in the Introduction, one might expect different resulting lattices or morphologies as two-dimensional diffusion rates are different between the substrates. Furthermore, prior investigations with S-layer proteins demonstrate that the charge and hydrophobicity of the substrate affect crystallization. On the other hand, one might argue that similar self-assembly behavior would be predicted since the length scales of typical surface forces would be much smaller than the thickness of the lipid bilayer. The electrostatic Debye length, for example, is estimated to be only ∼1.5 nm at 50 mM NaNO3,42 while typical phospholipid bilayers are ∼5 nm thick;43,44 other surface forces, such as van der Waals interactions, would be expected to operate over even shorter lengths. This similarity between crystallization behavior at the two different surfaces suggests that the latter expectation prevails and the local intermolecular interactions between proteins are more dominant than interactions between the substrate and protein. Earlier studies have also concluded that, in a mica-supported streptavidin system, local electrostatic interactions on the surface of proteins can contribute significantly to affect long-range interactions, steer supramolecular assembly, and modulate overall surface properties.45 Furthermore, our data indicates that the differences between diffusivities at the air-water28,29 and liquid-solid30–32 interfaces do not play a large role in determining the molecular configuration in this particular system. (42) Israelachvili, J. Intermolecular and Surface Forces; Academic Press: London, 1992. (43) Wiener, M. C.; White, S. H. Biophys. J. 1992, 61(2), 434–447. (44) Buldt, G.; Gally, H. U.; Seelig, A.; Seelig, J. Nature 1978, 271(5641), 182–184. (45) Leckband, D. E. AdV. Biophys. 1997, 34, 173–190.

While the exact source of the differences between crystallization at the air-water and solid-liquid interfaces cannot conclusively be identified through these investigations, it is likely due to either the presence of the solid substrate or the lipid composition modifications made to the supported bilayers. In the latter case, these changes in experimental conditions were necessitated by the presence of the solid substrate. For example, while the filler lipid is DOPC in air-water experiments, this lipid did not yield a stable second leaflet in the solid-supported bilayer, presumably due to its relatively flexible structure. Consequently, DTPC, with its saturated fatty-acid carbon chains, was used instead. d. Effect of Surface Ligand Concentration on Assembly. In addition to formation of C222 and a new P1 crystal, as described above, we also observed the type of P1 crystal which had previously been reported at the air-water interface for pH 4 (see Table 2). This molecular arrangement was obtained on the solid mica substrate only at relatively low surface ligand (biotin) concentrations under neutral pH conditions, and its dependence on concentration was explored in a previous manuscript.32 What is particularly notable is that these needle-like crystals formed even with SA-R (13-139), which had not been observed previously for this form at the air-water interface. Data in that investigation pointed to the streptavidin tetramer’s flexibility (relative to the crystallization plane) at low concentrations as enabling formation of the P1 crystal. e. Effect of Ionic Strength on Crystal Size and Kinetics. One desired end point in our studies is to grow relatively large, highly arrayed functional protein monolayers with low levels of molecular defects and understand how the size and quality of these crystals can be controlled. In past studies, streptavidin crystals were first grown at the air-water interface and then transferred to solid substrates such as HOPG46 or carbon-coated electron microscope grids17 for molecular characterization. While single crystals at the interface prior to transfer can be several hundred micrometers along a single dimension,47 the transfer process fractures these arrays, resulting in crystals that are tens to a few hundred nanometers in width. In contrast, growth of protein crystals directly onto solid substrates have yielded crystalline domains which are on the length scale of micrometers41 to hundreds of micrometers.31,32 Protein crystal morphology, crystallization kinetics, and molecular lattice parameters have been found to be highly dependent on the ionic strength of the buffers.48–50 In fact, changes (46) Scheuring, S.; Muller, D. J.; Ringler, P.; Heymann, J. B.; Engel, A. J. Microsc. 1998, 193, 28–35. (47) Wang, S.-W. Effects of Interaction Modifications in Two-Dimensional StreptaVidin Crystallization; Stanford University: Stanford, 1999. (48) Bhamidi, V.; Skrzypczak-Jankun, E.; Schall, C. A. J. Cryst. Growth 2001, 232(1-4), 77–85. (49) Gallagher, D. T.; Pan, Q. W.; Gilliland, G. L. J. Cryst. Growth 1998, 193(4), 665–673.

Molecular Self-Assembly of Solid-Supported Protein Crystals

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Figure 6. Effect of ionic strength on crystals grown to confluency on a solid substrate at 5% biotin-X-DHPE/95% DTPC at pH 7. (A) Crystals grown at 5 mM NaNO3. (B) Crystals grown at 50 mM NaNO3. (C) Crystals grown at 500 mM NaNO3. Scale bars are 50 µm.

in ionic strength for two-dimensional streptavidin crystallization at the air-water interface have been reported to yield needlelike P1 crystals or X-shaped C222 crystals, depending on salt concentrations under special conditions.16 We therefore investigated the effect of electrolyte concentration in our solidsupported system. We fixed the lipid composition at 5% biotin-X-DHPE/95% DTPC and measured the time required to reach confluency at different salt concentrations. Due to the higher homogeneity of recombinant systems, these experiments were performed using SA-R. Representative images of the resulting crystalline surfaces are presented in Figure 6. We observe that overall time for crystal growth is strongly dependent on ionic strength, significantly increasing as ionic strength is decreased. At salt concentrations of 5, 50, and 500 mM NaNO3 at neutral pH, crystals are confluent after 7, 4, and 0.75 h of incubation, respectively. This effect on growth kinetics is not unexpected as increases in ionic strength will decrease the Debye length and screen electrostatic repulsion between protein molecules.42 Under these conditions, the effect of this electrostatic screening appears to play a greater role in crystal growth than the corresponding variations in mass transport. Using FRAP, we measured the diffusion coefficient of surface-supported streptavidin at 50 and 500 mM NaNO3 and obtained values of 1.9 ( 0.1 × 10-8 and 1.5 ( 0.1 × 10-8 cm2/s, respectively. Although transport of protein to the crystal edge is slower at higher ionic strengths, the crystal growth rate at these concentrations is much faster. This indicates that collisions between a protein molecule to the edge of the crystal at these highly screened concentrations are more “productive” and likely to result in an addition to the crystalline lattice. Mechanistically, the decrease in electrostatic repulsion enables proteins to interact more closely and consequently increases the possibility of favorable collisions that result in crystallization. Although crystallization kinetics are increased with increasing ionic strengths, our AFM data demonstrate that there is no effect on lattice parameters. For the salt concentrations tested in the range of 5-500 mM, all crystals displayed C222 symmetry. While this appears to contradict air-water investigations in which P1 and C222 were observed at high and low salt conditions, respectively, there are differences in experimental systems.16 One important variance is the inclusion of sucrose in the solution phase of prior work. Disaccharides can interact with protein molecules by modulating hydrogen bonds,16,51 and this type of interaction, together with the electrostatic changes through salt conditions, could account for the discrepancy. Furthermore, the (50) Kubota, T.; Homma, K.; Noda, J.; Yamane, T.; Ataka, M. J. Cryst. Growth 2001, 233(4), 813–822. (51) Lopez-Diez, E. C.; Bone, S. Biochim. Biophys. Acta, Gen. Subj. 2004, 1673(3), 139–148.

reported air-water investigation used the native form of streptavidin (SA-N), while our investigations here use recombinant streptavidin (SA-R). One important observation is that single domains grown in low ionic strength are larger than those grown in high ionic strength (Figure 6). Domain lengths of a single crystal at 500 mM are on the order of ∼100 µm, while single crystals at low salt concentrations (5 and 50 mM) were typically 3-5 times larger along a single dimension. This effect can be explained by nucleation rates; Baird and Kim extended classic nucleation theory to include charge complexities in protein molecules and found that nucleation rates increase with increasing electrolyte concentrations for protein crystals.52 Similar to increases in the crystalline growth rates, this increase in nucleation is due to the screening of Coulombic repulsion forces. It is apparent from fluorescent microscopy images, however, that the macroscopic quality of these crystals is lower at 5 mM than at 50 or 500 mM. Figure 6A shows that within the singlecrystalline domains, at low ionic strength, narrow noncrystalline channels can be observed. These defects in the crystal can also be accounted for by the decreased screening at the low salt concentration: either molecules cannot interact as closely together to form optimal contacts within the crystal or they are more fragile and prone to dissociate from the bulk crystal. Therefore, to obtain an “optimal” balance of characteristics desired in device fabrication (such as large single-crystalline domains with low molecular defects), an intermediate ionic strength should be used. In a recent review describing use of the second osmotic virial coefficient to predict protein crystallization, Tessier and Lenhoff noted that there are four main patterns in which this coefficient can be a function of ionic strength.53 The second osmotic virial coefficient refers to a thermodynamic parameter which describes pairwise protein-protein interactions, and the intermediate range of values in which crystals are formed is known as the “crystallization slot”.54 Since the interactions between streptavidin molecules appear to be repulsive at low ionic strength and attractive at high ionic strength and since these proteins can be crystallized over a wide range of pH values, our data suggest that streptavidin’s behavior is consistent with Derjaguin-LandauVerwey-Overbeek (DLVO) theory. f. Functionality of Solid-Supported Streptavidin Crystals. To probe whether the crystalline monolayers are functional, biotinylated gold nanoparticles were incubated with C222 streptavidin arrays. We expect that the symmetry of the streptavidin tetramer will allow one face of the molecule to attach to the mica substrate while still enabling the opposite face to be (52) Baird, J. K.; Kim, Y. W. Mol. Phys. 2002, 100(11), 1855–1866. (53) Tessier, P. M.; Lenhoff, A. M. Curr. Opin. Biotechnol. 2003, 14(5), 512– 516. (54) George, A.; Wilson, W. W. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 361–365.

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crystals grown on mica.41 In those studies, liposomes adhered to the defect areas rather than the ordered molecules within the intact protein crystals. One possible explanation for this discrepancy could be the presence of strong lipid-lipid interactions between the vesicle particles and exposed lipids in the defective regions. In another investigation, nanoparticles were bound to a two-dimensional crystalline lattice which was grown at the air-water interface and transferred to a solid substrate.55 Unlike our coupling strategy, however, DNA hybridization was used as an additional linkage to attach the nanoparticles to the crystals.

Conclusions

Figure 7. Streptavidin crystal on mica after incubation with 20 nm gold nanoparticles functionalized with (A) biotin (BPT and 11-MUDA) or (B) no biotin (11-MUDA only). The difference in particle loading is a result of streptavidin’s specific affinity for the biotin ligands on the surface of the functionalized particles. AFM scan size is 2 µm.

available for binding nanoparticles. Figure 7 presents AFM images of these results and represents dark regions as bare mica, bright circular spots as 20 nm gold particles, and regions of intermediate intensity as protein crystals. They show that, as expected, nanoparticles which are modified with biotin will bind to the surfaces which contain protein crystals (Figure 7A). When nanoparticles are not coupled to biotin, there is relatively little nonspecific binding observed (Figure 7B). These results demonstrate that the crystalline regions are indeed functional and support the potential for using these protein crystals as templates for nanoscale patterning or materials assembly. Although no periodicity corresponding to the underlying lattice is observed, this is not unexpected; prior investigations with S-layer proteins showed that nanoparticle sizes need to be on the order of unit cell lengths for periodicity to occur.9 Furthermore, our observations suggest that particle binding does not appear to disrupt the underlying crystals. The molecular ordering and structural nature of the crystalline streptavidin monolayer present an additional level of complexity and stringency to the system that is not present for most streptavidinfunctionalized surfaces. For example, it was not known a priori whether the incubation with and attachment of relatively large, biotinlyated nanoparticles onto the crystal would cause crystal fracture or streptavidin dissociation (for example, due to competition of the biotin-binding sites). A limited number of other investigations have examined the binding functionality of two-dimentional streptavidin crystals. The results we present here are in contrast to previous experiments in which biotinylated liposomes were applied to streptavidin

In this investigation we show that the C222 and P1 crystal forms of streptavidin seen at the air-water interface can also be generated at the liquid-solid interface. This can be accomplished by varying solution pH, substrate composition, and genetic variant of the protein. Furthermore, we identified conditions which result in a new molecular configuration (also with P1 symmetry). We expected to observe crystals with P2 symmetry at low pH with the recombinant protein, and while our data is consistent with the growth of this crystal form, results are inconclusive due to the fragility of the crystal. Since the streptavidin variant can significantly affect self-assembly, we demonstrate the importance of determining the protein form and obtaining it from a consistent commercial source. Given the prevalence of streptavidin use in surface-binding applications, this finding could be particularly relevant to broader studies. Overall, the extent of similarity between crystallization behavior at the air-water and liquid-solid interfaces is large, demonstrating that the presence of the solid substrate does not appear to significantly alter two-dimensional crystal lattices. This can be explained by the length scales of typical surface forces since the penetration depth of these interactions would be much smaller than the thickness of the lipid bilayer. This suggests that local intermolecular interactions between protein surfaces play a larger role in crystallization than interactions between the interface and protein. By screening electrostatic repulsion between protein molecules we also demonstrate that we can modulate the quality, size, and growth rate of crystalline domains. However, the lattice configuration does not appear to be affected under the conditions examined. These crystalline domains were also found to be functional and could bind biotinylated gold nanoparticles. Our ability to manipulate molecular configuration, crystalline defects, and domain size on a functional array supports the potential of this system as a platform for organized materials assembly. Acknowledgment. We thank Pat Burroughs (Prozyme) for providing characterization data for their streptavidin product, Merce` Dalmau for performing mass spectrometry on our protein samples, and Daniel Mumm for helpful discussions regarding AFM characterization. This work was supported by the American Chemical Society-Petroleum Research Fund and the National Science Foundation (ECS-0609195). LA8004008 (55) Zou, B.; Ceyhan, B.; Simon, U.; Niemeyer, C. M. AdV. Mater. 2005, 17(13), 1643–1647. (56) Yatcilla, M. T.; Robertson, C. R.; Gast, A. P. Langmuir 1998, 14(2), 497–503. (57) 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.