Stimuli Responsive Hierarchical Assembly of P22 Virus-like Particles

Article Cite This: Chem. Mater. 2018, 30, 2262−2273

pubs.acs.org/cm

Stimuli Responsive Hierarchical Assembly of P22 Virus-like Particles William M. Aumiller Jr.,† Masaki Uchida,† Daniel W. Biner,† Heini M. Miettinen,‡ Byeongdu Lee,§ and Trevor Douglas*,† †

Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States Department of Microbiology & Immunology, Montana State University, Bozeman, Montana 59717, United States § X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439, United States ‡

S Supporting Information *

ABSTRACT: Biomimetic systems responsive to environmental stimuli are of growing interest due to their useful, intriguing, and sometimes unexpected properties. Hierarchical self-assembly of biological building blocks has emerged as a powerful means of creating biomaterials with collective properties. Here, we show P22 virus-like particles (VLPs) functionalized with a spider silk protein derivative on the capsid exterior assemble into a hierarchical structure due to spider silk/spider silk interactions at low pH and reversibly dissemble upon raising the pH. We also show that the capsid arrays can be assembled through electrostatic interaction between the negatively charged capsids and a positively supercharged GFP mutant and can be reversed by raising the ionic strength. Most notably, we found that the supercharged GFP could bind to the hierarchically assembled material under high salt conditions but not to the individual capsids under the same high salt conditions. The binding of the charged macromolecule under high salt conditions demonstrates a collective behavior of the hierarchically assembled system that is not present with the unassembled, individual components of the system.

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INTRODUCTION Hierarchical assembly to form novel materials has emerged as a prominent and diverse area of research because of the intricate and complex structures that are possible from single, modular building blocks.1−3 Virus-like particles (VLPs) are a prime choice for building blocks in higher order assembly because of their ability to self-assemble from multiple copies of just a few proteins to form individual capsids with well-defined symmetry and structure.4−7 The capsids can be genetically or chemically modified on both their interior or exterior surfaces,7−11 and many types of valuable cargo can be encapsulated within them.11−14 Furthermore, hierarchical assembly of the individual capsids into arrays has been achieved, demonstrating controlled higher order self-assembly.15−17 One overarching goal of synthetic biomimetic materials is to create hierarchical assemblies that respond to external stimuli by changing physical or chemical structure in response to environmental cues.18,19 This has been demonstrated for stimuli such as pH,20 ionic strength,16 temperature,21 and exposure to light.22 Typically these materials are only responsive to a single stimuli. Much work has been done developing polymers that respond to these stimuli as well.23,24 Specifically, a number of pH responsive polymers, also called intelligent or smart polymers, have been reported,20,25 and many of the same principles of design could be used for hierarchical biomaterials design of responsive systems. Nature provides us with many examples of individual components coming together to form assembled structures © 2018 American Chemical Society

that display new, nonadditive, or collective properties that are not found with the individual members.26 This is demonstrated on different biological length scales, such as many molecules coming together to form cells,27 many different specialized cells forming tissues/organisms,28 and many organisms forming ecosystems.29 For example, at the tissue level, skeletal muscle is composed of bundles of muscle fibers which are large cells formed during development from the fusion of many individual cells (myoblasts).30 In another example at the cellular level, some microbes form communities known as biofilms that are distinct from their individual (or planktonic) counterparts and offer advantages for the microbes.31−33 The biofilm provides increased defense against stressors (i.e., nutrient deprivation, pH changes, and antibiotics) and a pool of communal resources and allows for symbiotic relationships.31−33 Viruses, too, are examples of hierarchical assembly at the molecular level individual coat proteins have very different properties than the assembled viruses, and they come together in a self-assembly process to form highly regular capsids in which nucleic acid can be packaged, transported, and protected.6,34,35 In this work, we construct P22 VLP capsids, modified by the exterior incorporation of spider silk proteins that undergo hierarchical assembly upon changes in solution pH and ionic strength, and we demonstrate the resulting collective behavior Received: November 27, 2017 Revised: March 13, 2018 Published: March 15, 2018 2262

DOI: 10.1021/acs.chemmater.7b04964 Chem. Mater. 2018, 30, 2262−2273

Article

Chemistry of Materials

Figure 1. Scheme for construction of spider silk protein functionalized P22 VLPs. (a) The fusion protein consisting of the decoration protein (Dec) shown as a trimer (green), the flexible linker region (blue lines), and the spider silk protein (beige). (b) A 2D depiction of a P22 VLP decorated with the Spider silk fusion protein. A maximum of 80 trimers can bind to the capsid exterior. (c) The capsids are assembled in response to changes in the solution pH or ionic strength. At high pH and high salt, the capsids are not assembled, and at low pH and low ionic strength, they are assembled. Supercharged GFP (green sphere) mediates the electrostatic assembly and binds to the preformed assembly under certain conditions. electrocompetent cells and plated on LB-agarose plates supplemented with the appropriate antibiotic. Isolated colonies were selected, and the cells were grown in LB medium with the appropriate antibiotic to maintain selection for the plasmid. Supercharged GFP,39 Spidersilk,36 and DecWT40 were each expressed and purified as previously described. P22 S39C. Overnight cultures of BL21 (DE3) cells containing the P22 S39C with wild type scaffold pET11a plasmid were grown and used to induce 1 L cultures of LB media at 37 °C. Expression of the P22 S39C and scaffold was induced at 37 °C with isopropyl β-Dthiogalactopyranoside (IPTG) at a final concentration of 0.5 mM when the cultures reached OD600 = 0.6 (mid log phase). Cultures were grown for an additional 3 h after induction. Cells were harvested by centrifugation (3700 × g, 20 min, 4 °C) and stored at −80 °C overnight. The next day, the cell pellet was resuspended in phosphate buffered saline (50 mM sodium phosphate, 100 mM sodium chloride, pH 7.5) with lysozyme, DNase, and RNase. The cells were lysed by sonication. Cell debris was removed by centrifugation (12000 × g, 45 min, 4 °C). P22 was further purified from the resulting supernatant by ultracentrifugation (45000 × g, 50 min, 4 °C) with a 35% (w/v) sucrose cushion. The resulting viral pellet was resuspended in phosphate buffer saline (PBS, 100 mM NaCl, 50 mM sodium phosphate pH 7.4). Next, the sample was purified using an S-500 Sephadex column (GE Healthcare Life Sciences) size exclusion column using a Biorad Biologic Duoflow FPLC at a flow rate of 1 mL/ min. Fractions containing correctly assembled P22 were pooled and ultracentrifuged (45000 × g, 50 min, 4 °C). Purity was checked on an SDS-PAGE gel. P22 GuHCl Wash and Expansion. Wash. Unless noted otherwise, ultracentrifuge settings were 45000 × g, 50 min, 4 °C. The resulting P22 procapsids were ultracentrifuged, resuspended in a small volume of PBS buffer, and diluted 10× with 0.5 M guanidine hydrochloride (GuHCl) in order to remove scaffold protein. After 2 h of incubation at 4 °C, the sample was ultracentrifuged and resuspended in buffer followed by diluting in 0.5 M GuHCl to ∼25 mL. This was repeated two additional times. The resulting P22 empty shells were ultracentrifuged and resuspended in a low salt buffer (10 mM sodium phosphate, pH 7.5, 1 mM NaCl) at a concentration of 1 mg/mL. Expansion. The sample was divided into 1 mL aliquots of 1 mg/mL into 1.5 mL centrifuge tubes. The tubes were placed in a water bath at 66 °C and removed after 20 min. Complete expansion was confirmed by running samples of empty shell form and the expanded form on an agarose gel. 6xHis-Dec-Spidersilk. The expression of Dec-Ss was the same as for P22, except that 0.2% L-arabinose was used to induce expression. The next day, the cell pellet was resuspended in PBS containing 10 mM imidazole. Following cell lysis as described above, the Dec-Ss was purified using a Roche complete His-tag purification column. The sample was loaded on the column at a rate of 1 mL/min. The Dec-Ss was eluted using a stepwise gradient of up to 500 mM imidazole. Samples were dialyzed with 10 mM sodium phosphate, pH 7.5, to

of the hierarchical assembly (Figure 1). The P22 VLP is an icosahedral capsid with triangulation number = 7 assembled from 420 individual coat proteins (CP) that forms a capsid that is 56 nm in diameter. A genetic fusion between a P22 Decoration protein (Dec) and the N-terminal region of a Spider silk protein (Figure 1a) was created. The Dec portion of the fusion protein binds to the expanded (EX) form of P22, thus modifying the capsid to display spider silk protein on the exterior (Figure 1b). The spider silk protein undergoes a monomer to dimer conversion in response to pH, forming a dimer below pH ∼ 6.4.36,37 We show that the capsids undergo reversible hierarchical assembly in response to changes in solution pH caused by spider silk/spider silk interaction between capsids. In addition, the capsids hierarchically assemble in response to changes in solution ionic strength when a positively charged protein (supercharged GFP, GFP(+36)) is added as an assembly mediator (Figure 1c). Most notably, the GFP(+36) binds to the preformed spider silk mediated hierarchical assembly at high salt concentration (500 mM NaCl) in stark contrast to the observed lack of interaction between GFP(+36) and the individual capsids at this ionic strength.

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EXPERIMENTAL SECTION

Materials. Electrocompetent Escherichia coli cells were purchased from Sigma-Aldrich or Lucigen. DNase, RNase, lysozyme, and Atto 647N maleimide dye were purchased from Sigma-Aldrich. All other chemicals were from Fisher Scientific or Sigma-Aldrich. Molecular Biology. The Spidersilk gene containing two His6 tags and a thioredoxin tag (pT7HisTrxHisNT) was obtained from Nina Kronqvist.36,38 To create the Dec-Ss construct, two internal Nco I restriction sites were mutated from the spider silk coding sequence without changing the amino acid sequence. The mutagenesis was carried out by PCR using the Affymetrix Change-IT multiple mutation site directed mutagenesis kit with mutant forward and reverse primers and the pT7HisTrxHisNT plasmid as template. After sequence confirmation, the mutated spider silk DNA was amplified using a forward primer containing a SalI-site and a GS linker and a reverse primer containing a Hind III-site. The PCR product was gel purified, digested with SalI and Hind III, and ligated into the SalI and Hind III digested pBAD 6xHis-Dec plasmid. The final construct was confirmed by DNA sequencing (Eurofins MWG Operon, Inc.). The DNA and amino acid sequences are given in the Supporting Information. The supercharged GFP plasmid (pET-6xHis-(pos36)GFP) was obtained from David Liu (Addgene plasmid no. 62937). Protein Expression and Purification. The P22 S39C with wild type scaffold, DecWT Dec-Spider silk, Spider silk, and supercharged GFP (+36 GFP) constructs were transformed into BL21 (DE3) 2263


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Figure 2. Characterization of Dec-Ss bound P22 capsids. (a) The Dec-Ss fusion protein was visible as a single band on an SDS-PAGE gel (left). After incubation of the P22 capsid with the Dec-Ss protein, the Dec-Ss decorated capsids were pelleted by ultracentrifugation and resuspended in fresh buffer to remove any unbound Dec-Ss. Protein bands corresponding to P22 CP and the Dec-Ss were visible (right). (b) MALS analysis of P22 EX capsids alone (red traces) and Dec-Ss bound P22 (blue traces). There was a clear shift in retention time for the Dec-Ss bound P22 EX capsid and increase in capsid MW corresponding to complete saturation of the tight binding sites with Dec-Ss. Size Exclusion ChromatographyMulti-Angle Light Scattering (SEC-MALS). Samples (25 μL at 1 mg/mL) were injected on an Agilent 1200 HPLC system at a flow rate of 0.7 mL/min and separated over a WTC-0200S size exclusion column (Wyatt Technologies). The buffer was 50 mM sodium phosphate, 100 mM NaCl, pH 7.2, with 200 ppm of NaN3 to inhibit bacterial growth. The detectors were a UV−vis detector (Agilent), a HELEOS multiangle laser light scattering (MALS) detector (Wyatt), and an Optilab rEX differential refractometer (Wyatt). The number-average molecular weight, Mn, was measured at fwhm of each peak with Astra 6.0.3.16 software using a dn/dc value of 0.185 mL/g. Before injection of the P22 Dec-Ss sample, any unbound Dec-Ss was first removed by ultracentrifugation. Zeta Potential. Zeta potential measurements were collected using a Malvern Zetasizer Nano-S instrument with a disposable folded capillary zeta cell. The Smoluchowski approximation was used to convert electrophoretic mobility to zeta potential. Confocal Microscopy and Capsid Labeling. Confocal microscopy images were taken using a Leica SP5 scanning confocal microscope. The P22 capsids were visualized by labeling at C39 with Atto 647N maleimide. For capsid labeling, the dye was added at a ratio of 10 per C39 and allowed to react for 5 min. The reaction was quenched by adding excess dithiothreitol. Free dye was removed by ultracentrifugation. Excitation wavelengths used were 488 nm for the GFP(+36) and 633 nm for the Atto 647N capsids. Images were analyzed using Fiji software. Transmission Electron Microscopy. TEM images were taken using a JEOL 1010 transmission electron microscope at an accelerating voltage of 100 kV. The samples (0.1 mg/mL, 5 μL) were applied to the carbon coated grids and incubated for 30 s. Excess liquid was wicked away with filter paper. Next, 5 μL of water was added and incubated for 30 s, and the liquid was wicked away, followed by incubation of 5 μL of uranyl acetate for 20 s and wicking away of the excess liquid. Small-Angle X-ray Scattering (SAXS) Measurement and Data Analysis. SAXS measurements of the hierarchically assembled P22 samples were performed at the 12ID-B beamline at the Advanced Photon Source (APS). The samples were measured at 13.3 keV, and the scattering data was collected with a Pilatus 2 M detector. The sample to detector distance was set as 3.6 m. The system was

remove the imidazole. The concentration of the Dec-Ss was determined by UV−vis absorbance readings at 280 nm, using a calculated extinction coefficient (24400 M−1 cm−1) from ExPASy. Supercharged GFP. Supercharged GFP was purified as described for 6xHis-Dec-Spidersilk except that the NaCl concentration in the buffers was increased to 2 M NaCl. Empty shell P22 EX Hierarchical Assembly with Dec-Ss Protein and GFP(+36). Capsids were added at a concentration of 0.1 mg/mL when no GFP(+36) was present or 0.4 mg/mL when GFP(+36) was present to a solution containing 25 mM sodium phosphate buffer, pH 7.2. The solution also contained 100 mM urea and the appropriate amount of Dec-Ss and NaCl (and DecWT and Ss for inhibition experiments). The increase in turbidity associated with hierarchical assembly of P22 was monitored using absorbance as a proxy for light scattering and was collected on an Agilent UV−vis at a wavelength of 500 nm (samples without added GFP(+36)) or 600 (for samples with added GFP(+36)) by taking a measurement every 10 s. The pH was monitored using a Mettler Toledo SevenCompact pH/ Ion meter S220 m with a microtip pH electrode from Microelectrodes, Inc., by taking a measurement every 10 s. Samples were prepared by adding all components and allowing ∼2 min for the Dec-Ss to bind the EX P22. The assembly was initiated by adding solid δ-glucono-lactone (10 mg for a 500 μL reaction) and, after complete solubilization, immediately placing into the cuvette. For reactions involving pH increase for disassembly, the reaction was initiated by adding 2 μL of 50 mg/mL urease to 500 μL of sample. For samples with GFP(+36), it was the last component added. The assembly was controlled either by dilution to lower ionic strength which causes hierarchical assembly or by addition of NaCl to raise ionic strength which cause hierarchical disassembly. Each hierarchical assembly condition was repeated at least 3 times. GFP(+36) Quantification. The GFP(+36) was added at a ratio of 200 molecules per capsid. After assembly, the amount of GFP(+36) in the assembly was determined by absorbance measurements of the GFP(+36) remaining in solution after assembly. The GFP(+36) extinction coefficient was calculated, and it was independent of the pH over the pH range tested. Aliquots of hierarchically assembled samples were removed and centrifuged to pellet the assembly, and the GFP(+36) remaining in solution was quantified; the amount in the assemblies was calculated by subtraction from the total amount added. 2264


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Figure 3. Reversible assembly/disassembly of P22 capsids via Dec-Ss. (a) Plot of both pH (left axis, black traces) and absorbance (right axis, red traces) as a function of time (bottom axis) for 1× concentration of Dec-Ss trimers (60 Dec-Ss trimers per 60 binding sites on each capsid). The capsid concentration was 0.1 mg/mL. The pH drops at a nearly constant rate until 15 min, at which time urease is added and the pH begins to increase (blue dashed line). The solution turbidity, measured as absorbance at 500 nm, increases as the pH is lowered, indicating assembly of P22 capsids. Raising the pH results in a drop in absorbance. Three separate trials are indicated by the solid lines, smaller dashed lines, and larger dashed lines. (b) Multiple cycles of capsid assembly/disassembly. The assembly/disassembly process is reversible and can be carried out for at least 3 cycles. The blue dashed lines denote when solution conditions have changed either by adding GdL (to lower pH) or by adding urease (to raise pH). (c) TEM images of the unassembled (left) and assembled (right) capsids. (d) Changes in absorbance of the solution as the pH is lowered with different ratios of Dec-Ss trimers. As the ratio of Dec-Ss trimers to capsid increased, the amount of scattering increased. (e) Changes in turbidity of the solution as the pH is raised. The ratios follow a similar trend as the assembly. calibrated using a silver behenate standard. Samples were probed with a 1 s exposure time, 2 s interval, and 20 total exposure shots per sample while they were continuously moved using a syringe pump system to minimize beam damage. The scattered radiation was converted into one-dimensional SAXS data through radial averaging of two-dimensional scattering patterns. A total of 20 SAXS data sets per sample were then merged to get the averaged profile. The background due to the sample buffer was measured separately and subtracted from the averaged data. The data were represented as scattering intensity I(q) as a function of scattering vector q: q=

modified the capsid exterior with a small protein derived from spider silk. We designed a bifunctional fusion protein consisting of a Decoration (Dec) protein that binds to the expanded P22 capsid and a spider silk protein. The C terminal of the Dec protein was extended to incorporate a flexible linker with Gly and Ser repeats, followed by the N-terminal (NT) domain of the spider silk (Ss) protein, major ampullate spidroin (MaSp) 1 from E. australis. The NT domain is sensitive to changes in pH, forming dimers below pH ∼ 6.5.36 Dec protein binds tightly as a trimer to the P22 expanded (EX) morphology of P22 at the quasi 3-fold sites (KD = 9.2 nM, 60 per capsid) and weakly at the true 3-fold sites (KD = 1502 nM, 20 per capsid).8 Each capsid has up to 80 sites for the Dec trimer; therefore, 240 monomers or 80 spider silk trimers can bind. Characterization of Dec-Spider Silk Fusion Protein. The bifunctional Dec-Spider silk fusion gene was successfully expressed and purified. The gene contained an N-terminal histidine tag (6xHis-Dec-GS linker-Spider silk, or Dec-Ss) and the DNA sequence was verified. The fusion protein was expressed in E. coli, purified using Ni NTA chromatography, and the presence and purity of the fusion protein was confirmed by SDS-PAGE (Figure 2a) and mass spectrometry (31350 Da observed, 31349.3 Da expected, Supporting Information Figure 1). The Dec-Ss fusion protein binds to the P22 EX form. A stoichiometric amount of Dec-Ss equivalent to occupancy of all tight binding sites was added to P22 EX. After binding, the capsids were purified by ultracentrifugation, the supernatant

4π sin θ λ

where θ is half of the scattering angle 2θ and λ is the X-ray wavelength used for the measurements. The overall X-ray scattering intensity I(q), which is measured experimentally, is a combination of form factor P(q) and structure factors S(q) with the following relationship:41 I(q) = kP(q)S(q) where the constant k is a factor related to the concentration of particles. Form factor P(q) is inherent to the sizes and shapes of individual P22 particles, and structure factors S(q) are inherent to the arrangement of these particles relative to one another. Because the arrangement of P22 particles in the assembled bulk material is the primary interest of this work, S(q) were extracted from I(q) data as described previously.42

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RESULTS AND DISCUSSION In order to create hierarchical assemblies of P22 capsids that exhibit stimuli responsive behavior to changes in pH, we 2265


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amount of added Dec-Ss was increased, the absorbance also increased, suggesting larger arrays formed (Figure 3d and Supporting Information Figure 3). Scattering was decreased at half site occupancy (0.5×) compared to 1× and minimally at 0× Dec-Ss. While the Dec-Ss fusions appear to maintain a trimeric form, this does not guarantee that the interparticle interactions between P22-Dec-Ss are perfectly head-to-head, and we considered the possibility of cross-linking/polymerization of the Dec-Ss trimers. We hypothesize that some crosslinking of the excess Dec-Ss allowed for unattached Dec-Ss trimers to be incorporated into the assembly, resulting in the formation of the larger P22 assemblies. Although the number of capsids in each assembly was the same, the density of capsids in the assembly would be decreased with increasing Dec-Ss under this mechanism (see Figure 6). The disassembly traces (Figure 3e) also show that for the higher ratios (5× and 10×) there was a nearly linear decrease in absorbance from pH 5.6 to pH 6.3 at which point the material completely disassembled. We attributed the loss of scattering to loss of connectivity among the spider silk within the cluster as some of the connections weakened. It has been shown upon initial dimerization of the NT spider silk at pH ∼ 6.5 that a further drop in the pH results in the formation of more strongly associated dimers.36 Therefore, raising the pH would cause weakening of the Ss−Ss interactions. We ruled out some other possibilities for the loss of scattering over this pH range. The decrease was not caused by the clusters settling to the bottom of the cuvette as lowering the rate of pH change by adding concentrated buffer (Supporting Information Figure 4) over the same time frame did not decrease the scattering. Additionally, it was not caused by weakening of the Dec− capsid interaction. We measured the amount of wild type Dec protein (DecWT) bound at pH 6.8 and 5.5 by SDS-PAGE/ densitometry, and there was no significant difference in the amount of DecWT bound (Supporting Information Figure 5). We also tested the 10× ratio of the Dec-Ss alone (Figure 3d,e), but at this ratio there was no significant increase in scattering over the pH range tested. Therefore, we concluded that the changes in turbidity are due to scattering from assembled capsids and not from Dec-Ss up to 10×. Both the 1× Dec-Ss and the 10× Dec-Ss mediated capsid assemblies show some long-range order and contain face centered cubic (FCC)-like closed packed domains. The structures of the higher order assemblies formed from the Dec-Ss modified capsids were analyzed with small-angle X-ray scattering (Figure 4). The structure factor of the assembled samples, modified with either 1× Dec-Ss or 10× Dec-Ss, showed peaks indicating some degree of ordering. The positions of the first two peaks of 1× Dec-Ss matched well with a simulated SAXS profile of a capsid array having an FCC structure with lattice parameter a = 103 nm, indicating that the assemblies are likely to contain FCC-like ordered domains. In the higher q region, the measured peaks were not well resolved and peak positions did not match well to a simulated FCC structure. This suggests that the majority of structures or the crystalline domains possess very short-range ordering. In addition, significant broadening of the peaks with increasing q also suggests liquid crystalline ordering; i.e., positions of particles keep translational symmetry but not angular symmetry.41 In other words, the interparticle distances are uniform but the packing structure, e.g., hexagonal, is not well developed. The peaks of the sample assembled in the presence of 10× Dec-Ss were sharper than those of the sample with 1×

containing any unbound Dec-Ss was removed, and the capsids were resuspended in fresh buffer. We used both size exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS) and SDS-PAGE to determine that the Dec-Ss was bound to the P22 EX. SDS-PAGE showed two bands, corresponding to the Dec-Ss fusion and P22 coated protein (CP) (Figure 2a). MALS analysis (Supporting Figure 1 Table 1) showed an increase in molecular weight of the Dec-Ss bound P22 (25.3 ± 0.6 MDa) compared to P22 EX (18.6 ± 0.1 MDa) that corresponds to full occupancy (71 ± 6) of the tight binding sites. These numbers agreed well with the predicted mass of each type: 25.3 MDa for the Dec-Ss P22 capsids and 19.6 MDa for P22 EX only. The Dec-Ss bound P22 EX also showed a shift to an earlier retention time in the SEC as compared to the EX (Figure 2b), and the increase in size was confirmed by a measured increase in the radius of gyration (Rg) upon Dec-Ss binding (29.2 ± 0.1 nm for EX P22 and 32.2 ± 0.1 nm for Dec-Ss-P22). Reversible Hierarchical Assembly/Disassembly of P22 Capsids and Analysis of the Assembled Structure. The spider silk modified capsids formed hierarchical assemblies when the pH of the solution was lowered while a subsequent increase of the pH resulted in disassembly (Figure 3a), indicating that the process was completely reversible. We used turbidity (light scattering) measurements of the solution at 500 nm to monitor higher order capsid assembly/ disassembly and simultaneously measured the solution pH with a microtip pH electrode. The P22 capsids were incubated with a stoichiometric equivalent (for occupancy of the tight binding site, 1×) of Dec-Ss in sodium phosphate buffer (pH 7.2, 20 mM). The controlled lowering of the pH of the solution was initiated by addition of solid δ-gluconolactone, which hydrolyzes to gluconic acid. This approach gradually lowers the solution pH at a nearly constant rate without changing the solution volume, unlike a titration with acid.43,44 At pH ∼ 6.2, there was a sharp increase in turbidity (scattering), indicating extensive interparticle interaction associated with higher order assembly of the P22 capsids. The turbidity eventually reached a plateau, presumably due to removal of all the free capsids in solution. In order to raise the pH to demonstrate reversibility of the hierarchical assembly, urea was included in the solution and at 15 min (pH ∼ 5.6) the enzyme urease was added. Urease hydrolyzed the urea to generate CO2 and ammonia, thus gradually raising the solution pH at a constant rate. The turbidity remained relatively constant from pH 5.6 to pH 6.1 and increased slightly from pH 6.1 to 6.4. At pH 6.4 there was a sharp drop in turbidity indicating that the arrays dissembled. The assembly/disassembly process was reversible for up to 3 cycles of alternating low and high pH (Figure 3b). Imaging of the assembled samples by transmission electron microscopy (TEM) revealed large clusters of capsids, which are quite distinct from the individual particle morphology seen in the unassembled samples (Figure 3c and Supporting Information Figure 2). The cluster sizes were very polydisperse, with an average size of 4.7 ± 3.0 μm. These data demonstrate the ability of the Ss decorated capsids to reversibly assemble and disassemble in response to solution pH, due to the multivalent connectivity afforded through the spider silk protein interactions between capsids. Addition of higher ratios of Dec-Ss resulted in an increase in the size of the hierarchical assemblies. We varied the amount of Dec-Ss added from no Dec-Ss up to 10× the number of tight binding sites (i.e., 600 Dec-Ss trimers added per capsid). As the 2266


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Decreasing the density of Dec-Ss on the exterior of the capsids provided another means to decrease size of the clusters (Figure 5c,d, Supporting Information Figure 7). The density of Dec-Ss was decreased by adding different ratios of Dec-Ss and DecWT, while keeping the total number of Dec trimers constant. Minimal cluster formation was observed at 25% DecSs, and the scattering decreased by more than half with 50% occupancy as compared to 100% Dec-Ss occupancy. As seen in the disassembly traces (Figure 5d), there was an increase in scattering between pH 6.0 and 6.4 before the decrease in scattering associated with complete disassembly at pH 6.4. Recall that we observed this effect with the Dec-Ss only at higher ratios, but the effect was more pronounced in these experiments than those with only Dec-Ss at higher ratios. The addition of Dec-Ss and DecWT together allowed for an interesting possibility where the trimers bound to the capsids could be consist with both Dec-Ss and DecWT. If just one or two of the Dec trimers contained Ss, then the number of headto-head, capsid-to-capsid connections would be fewer and could provide more flexibility in the assembly as the connections weakenedallowing for the assemblies to “swell” and increase in size before complete disassembly (see Figure 6c). The capsids were still able to form hierarchical assemblies, even when most of the tight binding sites were blocked with DecWT (Figure 5e,f, Supporting Information Figure 6). The tight binding sites were first saturated with 5× DecWT. Dec-Ss was subsequently added, at a ratio of 5×, to occupy any remaining sites. Under these conditions of DecWT and Dec-Ss, all 60 of the tight binding sites and about 13 of the 20 weak binding sites should have been occupied by DecWT. On average just 3 of the remaining binding sites would have been occupied by the Dec-Ss. Since the amount of scattering was nearly as large as when the Dec-Ss and DecWT were added simultaneously, it suggested that the free Dec-Ss, not bound to the capsids, was facilitating the assembly. The Dec-Ss bound to the capsids could form connections with the Dec-Ss trimers that were free in solution. Since only one monomer of the DecSs trimer would be necessary for bonding with another monomer on another capsid, the remaining two monomers could polymerize with other Dec-Ss trimers free in solution. Based on these data, we propose a model for assembly of the capsids (Figure 6). When there is a nearly stoichiometric amount of Dec-Ss to the tight binding sites (Figure 6b), the capsids make a number of direct connections with their neighbors. Therefore, upon raising the pH, there is less change in assembly size from weakening connections. While the concentration of P22 EX is fixed and the concentration of DecSs (or free spider silk, Figure 6c) is increasing, the unbound spider silk becomes part of the assembly and results in larger arrays. When the pH is raised after assembly, the connections are weakened. Thus, losing some of the connections could result in loss of capsids from the outer part of the clusters and a decrease in cluster size until the clusters completely disassemble. In the cases where DecWT is added together with the Dec-Ss at an amount equivalent to the tight binding sites (Figure 5c,d), the Dec-Ss on the surface of the capsid is less dense. As some of the connections are weakened/broken by raising the pH, the few remaining connections allow for more flexibility in the assembly due to the long flexible linker between Dec and Ss (25 amino acids). Thus, the clusters begin to unravel and grow larger in size before completely disassembling (Figure 6d).

Figure 4. SAXS analysis of P22 capsid assemblies mediated by dimerization of Dec-Ss bound to adjacent P22 particles. SAXS data are plotted as structure factor S(q) versus scattering vector q. The simulated scattering patterns are generated from two FCC structures. Both structures have the same lattice parameter a = 103 nm and the nearest neighbor distance between capsids of 72.8 nm. However, relative orientations of icosahedral capsids in the lattices are different; i.e., they are oriented all face-to-face fashion (oriented) or random (nonoriented). The measured profile of the 1× Dec-Ss resembles more closely the simulated scattering pattern of the oriented lattice (for example, more significant 200 reflection than 111 reflection), whereas the measured profile of the 10× Dec-Ss resembles the simulated pattern of the nonoriented lattice.

Dec-Ss, suggesting the array of the P22 capsid assembled with 10× Dec-Ss has larger domain sizes. This could be because the incorporation of excess Dec-Ss as a linker provides more flexibility to the structure and allows the assembled material to anneal into a more well-ordered structure. Inhibition of Capsid Assembly with Free Ss and DecWT. The hierarchical assembly could be partially inhibited at low concentrations of free NT spider silk; however, the assembly size increased at high concentrations of free NT spider silk (Figure 5a,b). Inhibition of the hierarchical assembly occurred at low ratios of free Ss to the number of tight binding sites (0.5× and 1×). At these low ratios, the free spider silk can dimerize with Dec-Ss and effectively block binding with other capsids. At higher ratios, there was actually an increase in scattering, similar to what we observed with the high Dec-Ss ratios. A control experiment where bovine serum albumin (BSA) was added at a concentration of 10× the number of tight binding sites had no effect on the assembly, indicating that the scattering is specific to free spider silk. We also observed that below ∼ pH 5.8, the spider silk formed some aggregate structures. Additionally, we saw evidence of aggregation when we added an extremely high ratio (100×) of Dec-Ss both with and without capsids. However, the aggregation due to Dec-Ss on its own at 10× ratio was minimal (Supporting Information Figure 6). The free spider silk N terminal domain has been reported to form large assemblies/fibers under some conditions around pH 6 as evidenced by light scattering and turbidity measurements.37,45,46 Therefore, the larger assemblies could be facilitated not only by the trimeric Dec-Ss fusions as discussed above but also by the presence of free NT spider silk protein itself. 2267


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Figure 5. Effect of free spider silk and DecWT on assembly formation. In all cases, the concentration of P22 was 0.1 mg/mL. (a) Assembly and disassembly traces (b) with varying ratios of free Ss added to 1× Dec-Ss capsids and the corresponding disassembly traces. Assembly (c) and disassembly (d) traces with varying ratios of Dec-Ss and DecWT. There was very little assembly at only 25% coverage with Dec-Ss. Assembly (e) and disassembly (f) of 5× Dec-Ss and 5× DecWT both added simultaneously and the Dec-Ss after the DecWT. The excess Dec-Ss in solution can still facilitate assembly of the capsids. Additional trials are given in Supporting Information Figure 7.

Capsid Assembly with Supercharged GFP. In addition to assembly based on pH, the capsids could be assembled into a higher order structure through directed electrostatic interactions by changing the solution ionic strength. The capsids are highly negatively charged (zeta potential for P22 EX −25 ± 15 mV at pH 7.2, −28 ± 17 mV at pH 5.8; P22 EX w/Dec-Ss −21 ± 11 mV at pH 7.2, −17 ± 12 mV at pH 5.8), and we used a supercharged green fluorescent protein (GFP) variant, engineered to theoretically contain 36 positive charges GFP(+36), to act as a positively charged assembly mediator. The GFP(+36) mediated the hierarchical assembly of P22 EX with just 10 GFP(+36) molecules per P22 EX capsid at neutral pH (pH 7.2) as indicated by the formation of a turbid solution; the amount of scattering increased with higher ratios (Figure

7a). For all further experiments, we used a ratio of 200 GFP(+36) molecules per capsid (to allow for ease of quantification of the GFP(+36) and visualization of the clusters) and Dec-Ss decorated P22 EX capsids. We measured the salt dependence of the assembly and found that the particles assembled up to a threshold of 125 mM NaCl (Figure 7b). Above this concentration of salt the ionic strength was sufficient to screen interactions between the Dec-Ss P22 and GFP(+36) and no interparticle interactions were observed. The GFP(+36) and P22 interaction at low ionic strength provided a second mechanism for the directed interparticle assembly, and we were able to control which mechanism (either GFP(+36) or Ss) was used to assemble the capsids independently in the same sample (Figure 7c). An assembly schema was devised (Figure 2268


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Figure 6. Models for changes in assembly sizes with increasing pH. (a) Model representation of the P22 EX and Dec-Ss. (b) 1× Dec-Ss case. Many direct connections results in a minimal change in size as the pH is increasing, before completely disassembling at >pH 6.4. (c) Excess Ss case. Since there are fewer direct connections and the excess Dec-Ss is involved in the cluster formation, the clusters can shed outer layer capsids before completely disassembling. (d) The mixture of the Dec-Ss and DecWT case results in less dense Dec-Ss coverage. As connections weaken, the remaining connections allow more flexibility and cluster size increases before disassembling.

Figure 7. Hierarchical assembly of capsids occurs not only with changes in pH but also ionic strength. (a) Turbidity (measured as absorbance at 600 nm) vs ratio of GFP(+36):P22 EX. (b) Salt concentration dependence of assembly of Dec-Ss P22 EX with Supercharged GFP at pH 7.2. The particles assembled at 125 mM NaCl and below. (c) Hierarchical assembly of capsids based on electrostatic interactions and pH induced spider silk dimerization. Cases: (1) High pH, high salt: no assembly; (2) high pH, low salt: assembled via GFP(+36); (3) high pH, high salt: no assembly; (4) high salt, low pH: assembly via Ss; and (5) high salt, high pH: no assembly.

7b) to establish the synergistic effects of these two modes of interparticle interaction. In stage 1 (high salt, high pH condition) no hierarchical assembly was observed. In stage 2, the sample was diluted, lowering the ionic strength below the threshold for hierarchical assembly, and the capsids were

observed to assemble. In Stage 3, salt was added to return to the original ionic strength, and cluster disassembly occurred. When the pH was lowered in stage 4, the capsids assembled, and when the pH was raised in stage 5, the interparticle clusters were observed to disassemble. 2269


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Figure 8. (a) Pathways for hierarchical assembly of the capsids by changes in ionic strength and pH. In pathway 1, the capsids are hierarchically assembled first by Ss-Ss (1a, indicated by the straight lines between capsids) and then by GFP(+36) (1b). In the other pathway, capsids are assembled first by GFP(+36) (2a) and then by Ss-Ss (2b). (b) The arrays visualized by confocal microscopy. GFP(+36) is found in each of the arrays, even in the high salt, low pH case (1a).

Capsids Assembly with Both pH and Ionic Strength. Both mechanisms of assemblySs-Ss dimerization and GFP(+36)-capsid interactionscould be activated at the same time by lowering the pH and lowering the ionic strength. Figure 8a illustrates the two possible pathways for obtaining this assembled material via Ss-Ss and GFP(+36). At the beginning of both pathways, the individual capsids were present at high pH and high salt (0), conditions in which there was no interparticle interaction and therefore no hierarchical assembly. Following pathway 1, the pH was first lowered to generate a Ss mediated assembly (1a), and the salt concentration was subsequently decreased by dilution to allow GFP(+36) interaction with the Dec-Ss P22, thus ensuring both Ss and GFP(+36) mediated assembly (1b). In pathway two, the order was reversed: first the salt concentration was decreased (2a) by dilution of the sample affording an electrostatically mediated assembly, followed by a decrease of the solution pH (2b), which initiated Ss mediated interparticle interactions. We monitored the turbidity and pH traces for each case as a function of time (Supporting Information Figure 8), and we visualized each of the assembly states by confocal microscopy (Figure 8b). For each pathway, hierarchical assembly mediated by both mechanisms was evident since reversing only the pH or

ionic strength alone did not cause disassembly (Supporting Information Figure 8). Disassembly of the material could only be achieved when both the pH was raised and the salt concentration was increased, thus disrupting both modes of interparticle interaction. SAXS analysis of the higher order assemblies prepared through the different pathways revealed that the overall structures of the assembled samples were similar and adopt an FCC-like closed packed structure regardless of the pathway (Figure 9). The SAXS peaks from samples 1b and 2b were more pronounced than those of 1a and 2a, suggesting that ordering of the sample and interparticle distance is better defined when both mechanisms were applied, i.e., Ss-Ss dimerization and electrostatic interaction between GFP(+36) and capsid. Peak positions of 1a (Ss-Ss dimerization only) are slightly shifted to lower q from the other samples, indicating that the interparticle distance with Ss-Ss dimerization is slightly longer than the other sample with only electrostatic interactions. When electrostatic interactions are turned on to mediate the assembly, the interparticle distances are marginally contracted. GFP(+36) Colocalization within the Hierarchical Assemblies. Surprisingly, the supercharged GFP was found 2270


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Table 1. Number of GFP(+36) per Capsid in Each of the Assembled Statesa sample

amount of GFP(+36) in the hierarchical assemblies (number of GFP(+36) molecules/ capsid)

Pathway 1: Ss-Ss Assembled, followed by GFP(+36) Assembled 1a 80 ± 12 1a, GFP(+36) added after 53 ± 5 hierarchical assembly 1b 52 ± 22 1b after Ss-Ss reversal 72 ± 9 1b after GFP(+36) 98 ± 3 reversal (200 mM NaCl) Pathway 2: GFP(+36) Assembled, followed by Ss-Ss Assembled 2a 100 ± 13 2b 100 ± 2 2b after Ss-Ss reversal 77 ± 17 63 ± 11 2b after GFP(+36) reversal (500 mM NaCl)

Figure 9. SAXS analysis of the higher order assembly of P22 capsids prepared in the presence of Dec-Ss and GFP(+36). The array formation was mediated either by dimerization of Dec-Ss (1a) electrostatic interaction between capsid and GFP(+36) (2a), or via the combination of both of these mechanisms (1b and 2b).

a

The number of GFP(+36) molecules initially added was 200/capsid.

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CONCLUSION P22 VLPs were hierarchically assembled in response to either pH (spider silk) or ionic strength (GFP(+36)). Both assembly approaches could be independently activated on the same capsid demonstrating a high degree of control over the higher order capsid assembly mechanism. The capsids themselves are the product of a directed self-assembly, which occurs in vivo after the production of sufficient coat protein and scaffold protein subunits. In addition, formation of pH competent P22 capsids are the result of another self-assembly where the Dec-Ss fusion protein binds with high affinity to symmetry specific sites only on the assembled P22 capsid to create individual P22-DecSs particles, which are the building blocks for the further hierarchical assembly into bulk materials. Taken together, this illustrates the ability to control assembly within this system across multiple length scales. Our experimental system demonstrates a collective behavior of the hierarchically assembled capsids. The GFP(+36) is incorporated and retained within the Ss assembled capsid lattice but exhibits no discernible interaction with individual P22 capsids under the same high ionic strength conditions. This is due to increased multivalency and/or increased local electrostatics in the Ss assembly compared to the individual capsids. The primary driving force is presumably increased entropy, as the once associated small counterions are released upon the GFP(+36) binding to the clusters of capsids. This modular approach to hierarchical assembly offers a powerful means to integrate desired elements into the assembled structures, thus creating a smart material responsive to multiple stimuli. We have demonstrated that the interior of the capsid is a rich environment for selective encapsulation of a wide range of cargos;10,11,14,47−51 therefore, incorporation of a valuable, functional cargo on the interior of the capsid should be possible without impacting either of the current assembly mechanisms. Additionally, other desired protein sequences can be presented on the capsid exterior by fusion to Dec protein to create chimeric particles with both Ss and the protein of interest. Other highly charged macromolecules can also be used in place of or in addition to the supercharged GFP to incorporate other polymers into the hierarchically assembled structures.

to colocalize in the hierarchical assembly not only under low salt conditions but also under high salt and low pH conditions (assembly state 1a in Figure 8b), which is in striking contrast to the behavior of the individual P22 capsids. Under high salt conditions individual Dec-Ss P22 capsids showed no interaction with GFP(+36). Yet, when hierarchically assembled into an array, via the Ss interactions at high ionic strength, the GFP(+36) was found incorporated into the P22 assemblies. Experiments where GFP(+36) was added only after the array had formed still showed incorporation of the GFP(+36), which excluded the possibility of a purely physical entrapment. The ability of the Ss-P22 material to bind and incorporate the GFP(+36) under high salt conditions as the hierarchically assembled material, but not as the individual unassembled capsids, demonstrates the collective behavior of the extended material. We postulate that this behavior arises from the increased local negative charge in the assembly vs the individual capsids. We quantified the amount of GFP(+36) remaining in the assembly. This was done by removal of the assembly from solution by centrifugation, using absorbance measurements to determine the amount of GFP(+36) remaining in solution, and subtracting from the total amount added to determine the amount incorporated (Table 1). In all cases, there was GFP(+36) in the assembly. The amount of GFP(+36) in the assembly was lowest for the 1b case (Figure 8, Table 1), and comparable amounts were found for the other three cases. The number of GFP(+36) molecules per capsid ranges from ∼50 to 100, or about 2.5−5 GFP(+36) per triangular face on the icosahedron of Dec-Ss P22. There was hysteresis in the two possible assembly pathways, as the amount of GFP(+36) retained between them was slightly different. We also measured the amount of GFP(+36) in the assemblies when one of the assembly mechanisms was reversed and found that the GFP(+36) was retained in those cases. Of particular interest was the reversal of 2b (Figure 8, Table 1) by addition of 500 mM NaCl. Even at this high salt concentration, in which the individual capsids do not hierarchically assembly via electrostatic interactions with the GFP(+36), ∼63% of the original amount of GFP(+36) was retainedagain demonstrating the collective behavior of the hierarchical assembled capsids. 2271


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b04964. Dec-Ss DNA and protein sequence; mass spectrometry of Dec-Ss, absorbance and pH measurements as a function of time; absorbance at a constant pH; measurement of Dec binding to P22 as a function of pH; P22 hierarchical assembly at high ratio of Dec-Ss; additional trials of DecWT and Dec-Ss assembly; pH and absorbance as a function of time for assembly pathways 1 and 2; and raw MALS data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(T.D.) E-mail: [email protected]. ORCID

William M. Aumiller Jr.: 0000-0002-9260-3575 Masaki Uchida: 0000-0003-0710-8834 Byeongdu Lee: 0000-0003-2514-8805 Trevor Douglas: 0000-0002-7882-2704 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the US Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering (DE-SC0016155). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. We thank the staff of the Light Microscopy Imaging Center, the Electron Microscopy Center, and the Nanoscale Characterization Facility at Indiana University for use of the instrumentation in their facilities. We thank Nina Kronqvist for kindly providing the spider silk gene.

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