Conditional Network Assembly and Targeted Protein Retention via

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Conditional Network Assembly and Targeted Protein Retention via Environmentally Responsive, Engineered #-roll Peptides Beyza Bulutoglu, Sarah J. Yang, and Scott Banta Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 04 Jun 2017 Downloaded from http://pubs.acs.org on June 4, 2017

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Conditional Network Assembly and Targeted Protein Retention via Environmentally Responsive, Engineered β-roll Peptides Beyza Bulutoglu, Sarah J. Yang, Scott Banta* Department of Chemical Engineering, Columbia University 500 W 120th Street, Rm 801, New York, NY, 10027, USA * Corresponding author, email: [email protected]

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ABSTRACT Stimulus-responsive biomaterials have applications in many areas of biotechnology, such as tissue engineering, drug delivery and bioelectrocatalysis. The intrinsically disordered repeat-in-toxin (RTX) domain is a conformationally dynamic peptide, which gains β-roll secondary structure when bound to calcium ions. A smart hydrogel platform was constructed by genetically fusing two rationally designed mutant RTX domains: first, a mutant peptide with hydrophobic interfaces capable of calcium dependent network assembly and second, another mutant which conditionally binds a model target protein molecule, lysozyme. This way, the calcium-induced control over the secondary structure of the β-roll peptide was exploited to regulate both the cross-linking and the lysozyme binding functionalities. The constructed biomaterial exhibited calcium dependent gelation and target molecule retention, and erosion experiments showed that β-roll peptides with a higher affinity for lysozyme produced more robust hydrogel networks. This work demonstrates the use of RTX domains for introducing two useful features simultaneously, network cross-link and target protein binding, and that the calcium-dependent regulation of these systems can be useful for controlling bulk self-assembly and controlled release capabilities.

KEYWORDS Allosteric regulation, β-roll domain, proteinaceous hydrogels, stimulus-responsive, lysozyme


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INTRODUCTION New functional biomaterials are needed to advance tissue engineering and drug delivery technologies.1-6 Some of the potential applications include cell entrapment and delivery7, where the gel formation is achieved after the subcutaneous injection of the hydrogel samples; cell culture8,9, where cells are embedded within the gels under certain conditions for the manipulation of the cell behavior; drug delivery10,11, where hydrogels form via different stimuli such as changes in the pH or temperature12,13; and enzymatic applications14-16, such as electrode modifications for biosensors and bioelectrocatalysis. In particular, there is an interest in stimulus-responsive proteins (e.g. FEK16photochemically activated17; short peptides forming α-helical coiled coils-pH sensitive18; elastin-temperature responsive), which can be incorporated into hydrogel building blocks and trigger self-assembly via different mechanisms. We have previously reported the use of the Block V repeat-in-toxin (RTX) domain of adenylate cyclase from Bordetella pertussis for stimulus-responsive hydrogel assembly.19 This intrinsically disordered peptide folds into a β-roll secondary structure upon binding to calcium ions.20 It requires a capping group at C-terminus for proper folding and can transition between its folded and unfolded states, in the presence and absence of calcium, respectively.21-24 Previously, the amino acid residues on the β-roll faces were mutated to leucines, as shown in Figure 1. It was demonstrated that this mutant, DLeu β-roll, can be used as a stand-alone crosslinking moiety, where the network formation can be controlled by calcium addition.19 In the folded conformation, this peptide has two hydrophobic faces composed of leucine residues, which lead to self-assembly. In other work, the same β-roll domain was evolved to conditionally bind to a specific target, hen egg white lysozyme, which served as a


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model target protein.25 The mutant 406 β-roll was found to bind lysozyme with midnanomolar affinity upon concatenation. The binding behavior between the mutant peptide and lysozyme can be controlled by adjusting the calcium concentrations. The 3-D structure of the wild-type peptide as well as models of mutant peptides are shown in Figure 1, along with the residues on their mutated β-sheet faces.

Figure 1. Estimated structures of wild-type, 406 and DLeu β-roll peptides along with their primary sequences. Side and top-down views are shown for each construct in the putative folded conformation. Bound calcium ions and the C-terminal capping group are shown in red and grey, respectively. Residues located on the faces are highlighted in yellow, yellow and purple; and green for WT, 406 and DLeu, respectively. All domains were rendered in PyMOL using PDB file 5CVW26 as the wild type baseline structure. Here, we present a new hydrogel platform constructed based on these two different β-roll mutants (Figure 2). This bi-functional, stimulus-responsive hydrogel can capture lysozyme on its surface or entrap the target within the assembled protein


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network; and both oligomerization and target-capturing properties can be controlled via calcium addition. A second construct was produced where the concatemer of 406 β-roll is replaced with the concatemer of wild-type β-roll (which has two orders of magnitude lower affinity for lysozyme), to better explore the interaction of these biomaterials with lysozyme. The wild-type peptide does not actuate self-assembly.19,27 Therefore, the crosslinking ability of both constructs utilizes the calcium-dependent hydrophobic driving force provided by the DLeu β-roll mutants.

Figure 2. Monomeric hydrogel building blocks and assembled networks. Upon calcium binding, both DLeu β-roll and 406 β-roll transition to their folded state. DLeu β-rolls cross-link resulting in network assembly. 406 β-rolls capture lysozyme resulting in target retention within the assembled network. N-terminal monomeric maltose binding protein


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(MBP) aided the expression and purification of hydrogel constructs and did not contribute to network assembly or lysozyme interaction.19,28,29

MATERIALS AND METHODS Plasmids and Chemicals. The wild-type block V RTX domain with its capping group was amplified out of pDLE9-CyaA as described before.19 Amylose resin and enzymes used in gene cloning were purchased from New England Biolabs. Oligonucleotides used for cloning experiments were synthesized by Integrated DNA Technologies. E. coli 5alpha cell line was purchased from Bioline. Terrific broth media for protein expression and FITC-labeled lysozyme were purchased from Thermo Fisher Scientific. Ampicillin sodium salt and isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from Gold Biotechnology. Amicon centrifugal filters were purchased from Millipore. Sodium dodecyl sulfate polyacrylamide electrophoresis gels (SDS-PAGE) and gel running buffers were purchased from Invitrogen-Life Technologies. All other chemicals were purchased from Sigma-Aldrich. Construction of Individual β-roll Domains and Hydrogel Plasmids. Construction of the hydrogel forming β-roll mutant (DLeu β-roll) and wild-type β-roll were described previously.19 Lysozyme binding β-roll mutant (406 β-roll) was selected from a randomized library against immobilized hen egg white lysozyme via ribosome display as described elsewhere.25 For individual characterization, each construct was cloned into pMAL-intein backbone (a gift from Dr. David Wood (Ohio State University)) via KpnI and HindIII restriction sites.


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For the MBP-DLeuβ-WTβ-WTβ-DLeuβ hydrogel construct, a previously constructed vector was used: pMAL-MBP-DLeuβ-MBP-DLeuβ.19 The second MBP was replaced by the concatemer of the wild-type β-roll via BamHI and SalI restriction sites. For the cloning of the second hydrogel construct (MBP-DLeuβ-406β-406β-DLeuβ), the concatemer of the 406 β-roll was inserted into the same backbone via the same sites. Resulting plasmids were transformed into 5-alpha cells. The primers used in these cloning experiments are given in Table S1. Expression and Purification of Individual β-roll Domains and Hydrogel Constructs. The protein primary sequences of all peptides are given in Table S2. Both hydrogel constructs as well as single β-roll components were expressed in 1 L of sterilized Terrific Broth supplemented with 100 µg/mL ampicillin, 2 g/L D-glucose and 10 mL overnight culture, at 37 °C. The cells were grown to an OD600 of 0.6 while shaking and protein expression was induced with 0.3 mM IPTG. Expression was carried out for 5-7 h at 37 °C. After cell harvest (via centrifugation at 5000 × g for 10 min), the single β-roll components were re-suspended in 50 mL of MBP column buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4). The cells were sonicated (with an ultrasonication probe in ice bath for 6 min (5 s on pulse and 2 s off pulse)) and centrifuged again at 15000 x g for 30 min to collect the supernatant, which was loaded to columns packed with amylose resin. After lysate loading, the columns were washed 3 times with MBP column buffer and incubated with 8 mL of intein-cleaving buffer (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4, 40 mM bis-Tris, 2 mM EDTA, pH 6.2) overnight at 37 °C. Following the incubation, cleaved proteins are eluted off the amylose columns via 25 mL of MBP column buffer and further purified via 16/10 Q FF ion


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exchange column (GE Healthcare). For this purification step, the following low-salt and high-salt buffers are used: 20 mM bis-Tris, 25 mM NaCl, 25 mM NaCl, pH 6.0 and 20 mM bis-Tris, 25 mM NaCl, 500 mM NaCl, pH 6.0. Pure fractions are combined and concentrated via 10 kDa molecular weight cutoff amicon filters for further analysis. To purify the hydrogel proteins, the cells were resuspended in 50 mL MBP column buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4) and amylose columns were used after cell lysis with an ultrasonication probe in ice bath for 6 min (5 s on pulse and 2 s off pulse). After sample loading, the columns were washed with 100 mL of MBP column buffer, followed by the elution of the hydrogel constructs via 25 mL of buffer containing maltose (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 10 mM Maltose, pH 7.4). Eluted proteins were concentrated via 50 kDa molecular weight cutoff amicon filters and buffer exchanged into size exclusion chromatography (SEC) buffer (50 mM Tris, 150 mM NaCl, pH 7.4). Concentrated protein samples were run on SEC column as the last purification step. Chromatography fractions were run on SDS-PAGE, pure fractions were combined, concentrated and buffer exchanged into 5 mM Tris, pH 7.4. SDS-PAGE analysis of the purified MBP-DLeuβ-WTβ-WTβ-DLeuβ and MBPDLeuβ-406β-406β-DLeuβ hydrogel constructs is shown in Figure S1. Isothermal Titration Calorimetry (ITC). The affinity of the DLeuβ peptide for lysozyme was analyzed using a MicroCal Auto-iTC200. 350 µM peptide and 3.5 mM hen egg white lysozyme were used. The lysozyme was dissolved in 50 mM Tris, pH 7.4, modified with 10 mM CaCl2 and dialyzed against the same buffer, overnight prior to ITC experiment. Titrations were conducted by 19 identical injections of 2 µL with duration of 4 s per injection and a spacing of 150 s between injections. 8 ACS Paragon Plus Environment

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Hydrogel Sample Preparation. 13.2 wt % hydrogel samples were prepared in a similar way for both constructs. The total volume prior to lyophilization was 250, 500 or 1000 µL (in 5 mM Tris, pH 7.4), depending on the experiment. The samples were supplemented with 50 mM CaCl2 or MgCl2. Prepared solutions were frozen overnight and lyophilized the next day for 24 hr. The lyophilized protein powder was resuspended in 1/10 of the original sample volume (in deionized water (DI) water or in DI water supplemented with 35 µM FITC-labeled lysozyme). The samples were left at 4 °C overnight for proper rehydration, after mechanical mixing and centrifugation. For the experiment demonstrated in Figure S3, 4 x 100 µL samples were prepared with MBP-DLeuβ-406β-406β-DLeuβ in the presence of 50 mM CaCl2. After proper rehydration and gelation, CU (acronym for Columbia University) was written on a petri dish by squeezing the sample through an 18-gauge needle. 100 µL of 100 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was added on top of the gels after the plate was stored at 4 °C for 3 months. FITC-Labeled Lysozyme Retention Experiments. 35 µM FITC-labeled lysozyme in 1 mL of 50 mM Tris buffer supplemented with 50 mM CaCl2 or MgCl2 was added to hydrogel samples in centrifuge tubes, resuspended in DI water. After an hour of incubation, the samples were washed for 5 rounds by replacing 900 µL of the buffer with fresh Tris buffer supplemented with CaCl2 or MgCl2. After the final wash step, the samples were scooped out with a spatula, placed on a thin glass slide and visualized under UV light.


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Hydrogel Erosion Experiments. 1 mL of 50 mM Tris buffer supplemented with 50 mM CaCl2 was added to hydrogel samples resuspended in 35 µM FITC-labeled lysozyme solution. Total protein release and FITC release were recorded over time by measuring the absorbance at 280 nm and the fluorescence at excitation/emission wavelengths of 490/520 nm. The experiments were performed in triplicate for each hydrogel construct. A calibration curve was obtained by reading known concentrations of FITC-labeled lysozyme solutions at 490/520 nm. The released FITC data were converted to amount of FITC-labeled lysozyme and percentage release was calculated by dividing the obtained amount by the initially loaded FITC-labeled lysozyme amount. At the end of the experiment, all contents in the tubes were dissolved via centrifugation and protein concentrations were recorded by measuring the absorbance at 280 nm. These values were used to calculate the percentage protein release over time. The total protein release data and FITC release data were fit to Ritger-Peppas equation via OriginPro software.30 Analysis of Hydrogel Pictures via ImageJ. ImageJ software was utilized to quantify the color spectrum of the hydrogel pictures taken under UV light. Protein sample areas were selected and histogram built-in macro function was used to obtain the histograms in red, green and blue. Network Disassembly Experiments. For the experiment demonstrated in Figure 5, 6 x 100 µL samples were prepared with MBP-DLeuβ-406β-406β-DLeuβ in the presence of 50 mM CaCl2. After proper rehydration in 35 µM FITC-labeled lysozyme solution and gelation, CU (acronym for Columbia University) was written on three petri dishes by squeezing the sample through an 18-gauge needle. The petri dishes were covered with 25 mL of 50 mM Tris, 25 mL of 50 mM Tris supplemented with 50 mM CaCl2 or with 25 10 ACS Paragon Plus Environment

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mL of 50 mM Tris supplemented with 50 mM EGTA. Pictures were taken at different times points to observe the change in the hydrogel appearances with time. Statistical Analysis. All experiments were performed in triplicates. Two tailed t-test analysis with Welch’s correction was performed for the total protein release and FITClabeled lysozyme release to compare the n and k values (fitted via the Ritger-Peppas equation) between two different hydrogel constructs (MBP-DLeuβ-WTβ-WTβ-DLeuβ vs. MBP-DLeuβ-406β-406β-DLeuβ). Two statistical significance values were considered: P = 0.01 and P = 0.05.

RESULTS AND DISCUSSION Preparation of Hydrogel Samples. Previously, individual hydrogel building domains were characterized via circular dichroism (CD) spectroscopy to investigate the calciumbinding responsiveness of the mutant peptides.19,25 CD analysis revealed that both DLeu β-roll and 406 β-roll responded to increased calcium concentrations in a similarly as compared to the wild-type β-roll peptide with affinities for calcium ranging between 0.9 and 1.2 mM. Previously, more than two orders of magnitude difference was observed in the affinity of the concatemer of the 406 β-roll and of the wild-type β-roll, for binding lysozyme.25 The binding affinities (quantified via ITC) of DLeu β-roll, the concatemer of wild-type β-rolls and the concatemer of 406 β-rolls are presented in Figure S1. The crosslinker DLeu β-roll did not demonstrate any affinity for lysozyme. Based on the difference in the binding affinities between the concatemers of wild-type and 406 β-rolls, it is


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expected that differences would be observed in the target interaction capabilities between the two hydrogel constructs: MBP-DLeuβ-WTβ-WTβ-DLeuβ versus MBP-DLeuβ-406β406β-DLeuβ, which were purified via amylose columns (Figure S2). In order to probe this proposition, 13.2 wt %, 100 µL samples were prepared for both hydrogel constructs, with either 50 mM CaCl2 or 50 mM MgCl2, the latter serving as a negative control, since magnesium does not induce β-roll peptide folding.31 This weight percentage was chosen to normalize the molar cross-linking content to the 4 wt % samples as described previously.19 Images of samples with calcium and magnesium are shown in Figure 3. Protein samples with magnesium were viscous solutions whereas samples prepared with calcium appeared as assembled networks and could be easily picked up from a microcentrifuge tube with a spatula (Figure S3). Figure S4 demonstrates the disassembly of the network formed by MBP-DLeuβ-406β-406β-DLeuβ / Ca++, upon addition of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), which chelates the calcium ions causing the hydrogel to return to a viscous liquid state.


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Figure 3. Pictures of protein samples taken after incubation with FITC-labeled lysozyme. Samples with Mg++ remained as viscous solutions whereas Ca++ samples formed hydrogel networks. The surface of MBP-DLeuβ-406β-406β-DLeuβ was covered by FITC, indicating the presence of lysozyme. Complete pictures are shown in Figure S5. The bright spots in the photos with magnesium are small bubbles. Target Retention on Hydrogel Surface. FITC-labeled lysozyme (35 µM in 1 mL 50 mM Tris buffer supplemented with calcium or magnesium) was added to the protein samples and incubated for an hour. Each sample was washed 5 times by replacing the buffer in the microcentrifuge tube with fresh Tris buffer supplemented with Ca++ / Mg++, thus discarding the unbound lysozyme molecules. The tubes were visualized under the UV light, and images taken after the 4th and 5th rounds of wash are shown in Figure S6. The samples with magnesium were thick, viscous solutions and they partially localized to the bottom of the microcentrifuge tube and did not fully dissolve. After the final wash step, the buffer was discarded and the protein samples were removed with a spatula, placed on a glass slide and visualized both under visible light and under UV light to detect the presence of the FITC label. As presented in Figure 3, only the hydrogel construct with the 406 β-roll domain was capable of forming enough interactions with the FITC-labeled lysozyme and retaining it on the hydrogel surface, as indicated by the green color. The other samples appeared blue under the UV light, a result of their intrinsic protein fluorescence. Pictures of MBP-DLeuβ-WTβ-WTβ-DLeuβ and MBP-DLeuβ-406β-406β-DLeuβ were analyzed via ImageJ software to determine the difference in the sample pixel intensities (Figs. S7 and S8). A gain of high intensity in green pixilation is observed for MBP-DLeuβ-406β-406β-DLeuβ/Ca++ as shown in Figure S7. This shift in the intensity was likely caused by the presence of FITC-labeled 13 ACS Paragon Plus Environment

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lysozyme molecules. A similar result was not observed in the case of the samples with magnesium (Fig. S8). Hydrogel Erosion and Target Release. In order to further investigate the lysozyme binding and entrapping capacities, protein target retention and erosion rate experiments were performed. Matched percentage hydrogels were prepared with calcium for both constructs. These lyophilized samples were rehydrated in FITC-labeled lysozyme solution, in order to examine the release characteristics of the lysozyme and to assess the hydrogel erosion rate. One mL of 50 mM Tris buffer supplemented with calcium was added to all hydrogel samples and the increase in total protein release and in the released FITC amounts were plotted over time for 48 hours (Figure 4). A faster protein release was observed for MBP-DLeuβ-WTβ-WTβ-DLeuβ, for both 25 µL and 50 µL hydrogel samples (Figure 4B). The detected protein amount corresponded to the sum of the eroded hydrogels and the lysozyme released from the hydrogels. This difference was most likely correlated with the faster release of lysozyme, which was entrapped within the hydrogels, since the two constructs had similar material characteristics, other than their affinity for the lysozyme molecules. Another possible explanation for this difference might arise from the ability of lysozyme to dimerize.32 This might have contributed to the overall stability of the MBP-DLeuβ-406β-406β-DLeuβ construct, thus resulting in prolonged erosion of the hydrogel.


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Figure 4. Protein release and hydrogel erosion experiments. (A) A schematic demonstrating the experimental set-up. (B) Total protein release over time. The absorbance at 280 nm was recorded over 48 hours for MBP-DLeuβ-WTβ-WTβ-DLeuβ and MBP-DLeuβ-406β-406β-DLeuβ with two different hydrogel volumes (25 µL and 50 µL). The error bars denote the SEM (n=3). (C) Total FITC release over time. The fluorescence at excitation/emission of 490/520 nm was recorded over 48 hours for MBPDLeuβ-WTβ-WTβ-DLeuβ and MBP-DLeuβ-406β-406β-DLeuβ with two different hydrogel volumes (25 µL and 50 µL) and these values were used to calculate the mass of lysozyme released. The error bars denote the SEM (n=3). (D) Percentage total FITC released over time. The error bars denote the SEM (n=3). The FITC release profiles showed a difference between the two constructs, where the amount of free FITC-labeled lysozyme in solution was higher for the MBP-DLeuβWTβ-WTβ-DLeuβ (Figure 4C), for both hydrogel volumes. The percentage of lysozyme released is shown in Figure 4D. 25 µL hydrogel samples eroded faster in the case of both constructs. The percentage lysozyme released by MBP-DLeuβ-WTβ-WTβ-DLeuβ was faster, in agreement with the higher affinity of 406β-406β for lysozyme compared to WTβ-WTβ. Physical entrapment of lysozyme within the hydrogel samples is also possible as indicated by the incomplete release of the initially captured lysozyme at the end of release experiments. At the end of the 48 hours, the hydrogels were visualized under the UV light as shown in Figure S9. The color difference suggested a better retention of the entrapped lysozyme by MBP-DLeuβ-406β-406β-DLeuβ. Release Kinetics. The released protein absorbance data and the percentage of FITC released data were fit via Ritger-Peppas equation30: ெ೟ ெಮ

= ݇‫ ݐ‬௡

Eq. 1


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where Mt is the amount released at time t, M∞ is the total amount loaded, k is a kinetic constant and n is the diffusion parameter describing the release mechanism. There are two different assemblies to be considered: the dissociation of the DLeu β-rolls resulting in the erosion of the network and the dissociation of the 406 β-rolls and FITC-labeled lysozyme resulting in FITC release. For both total protein release and FITC release data, the fitted diffusion parameters appeared to be too low to indicate Fickian diffusion characteristics (n = 0.5, Table 1). For biodegradable polymers, the protein or drug release behavior can be governed by several mechanisms such as swelling, diffusion and erosion.33 A diffusion parameter close to 1 would indicate a primarily swelling controlled release. It is likely that the mechanism of release from these hydrogels primarily results from the network erosion, although the diffusion of the lysozyme from the hydrogels does likely contribute to the overall release. However, the interaction between the encapsulated lysozyme and individual hydrogel domains, especially 406β-406β, should reduce the contribution of lysozyme diffusion. Table 1. Release characteristics obtained via Ritger-Peppas model Construct / Hydrogel Volume

Total Protein Release

FITC Release

n

k

R2

n

k

R2

0.22 ± 0.01

0.068 ± 0.002

0.985

0.21 ± 0.02

0.056 ± 0.003

0.954

MBP-DLeuβ-406β406β-DLeuβ / 25 µL

0.30 ± 0.02**

0.020 ± 0.001*

0.966

0.032 ± 0.01*

0.033 ± 0.003*

0.960

MBP-DLeuβ-WTβWTβ-DLeuβ / 50 µL

0.28 ± 0.01

0.078 ± 0.003

0.988

0.33 ± 0.01

0.033 ± 0.001

0.992

MBP-DLeuβ-406β406β-DLeuβ / 50 µL

0.41 ± 0.03**

0.021 ± 0.001*

0.977

0.41 ± 0.02**

0.020 ± 0.001*

0.987

MBP-DLeuβ-WTβWTβ-DLeuβ / 25 µL


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The k values have units of hr–n. All values are reported as mean (n = 3) ± SEM. Parameters obtained for MBP-DLeuβ-406β-406β-DLeuβ were compared to the ones of MBP-DLeuβ-WTβWTβ-DLeuβ and statistically significant differences are denoted with * for P < 0.01 and ** for P < 0.05 (t-test).

The supramolecular hydrogels were composed of three components, the β-roll peptides, calcium ions and lysozyme. By keeping the Ca++ binding mechanism and DLeu β-roll interactions the same and changing the interaction of the lysozyme component, we explored the total protein release versus the lysozyme release. The affinity between lysozyme and 406 β-roll affects the overall release mechanism of the networks. The differences in the n values for the different constructs might be due to changes in the diffusivity. 406 β-rolls might still be bound to the lysozyme even after the bonds between the DLeu β-rolls break, reducing the overall diffusivity of the monomer. The kinetic constants were found to be larger for the constructs involving wildtype β-roll independent of the hydrogel volume, indicating a faster release of looselybound lysozyme. For MBP-DLeuβ-WTβ-WTβ-DLeuβ, larger k values were obtained for the total protein release, compared to FITC-labeled lysozyme release, while the n values did not change significantly. This suggests that the difference in the total protein release is due to faster erosion of the network in addition to the lysozyme diffusion. In addition, for MBP-DLeuβ-406β-406β-DLeuβ, both the n and k values are more similar between FITC release and total protein release, which suggests a better cross-linking within the network. As shown in Figure 4B, the total protein release was higher for the samples containing the wild-type peptide over the recorded time frame. The differences between


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the release characteristics are more prominent in the case of the total protein release compared to FITC-labeled lysozyme release (Figures 4C, D). The retarded total protein release in the samples with the mutant 406 β-roll might be due to the fact that the lysozyme bound to mutant β-rolls stabilizes the hydrogel network, slowing the release of the β-rolls in addition to the lysozyme. These results demonstrate how the use of low affinity (wild-type β-roll peptide) versus high affinity components (406 β-roll peptide) can impact the protein release profiles. These findings chart a path forward for tuning the noncovalent interactions in a protein hydrogel network enabling different release profiles. This 3-component system can be utilized in tunable controlled release experiments where the 3rd component could eventually be a therapeutic protein or other molecule. Demonstration of Control Over Network Assembly. Hydrogel samples with MBPDLeuβ-406β-406β-DLeuβ were prepared in the presence of calcium and the lyophilized protein samples were resuspended in FITC-labeled lysozyme solution. The letters CU were written with the hydrogel samples on three different petri dishes (Fig. 5). In order to assess the reversibility of calcium dependent gelation, the plates were covered with buffer (50 mM Tris), buffer supplemented with 50 mM CaCl2, and buffer supplemented with 50 mM EGTA. The hydrogel in the presence of buffer supplemented with calcium stayed intact where as the ones with buffer only and buffer with EGTA fell apart following the diffusion or chelation of the calcium ions away from the β-rolls. Tunable biomaterial release could likely occur with the intermediate calcium concentrations.


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Figure 5. Reversibility of the calcium dependent network assembly. Top panel shows the pictures of plates covered with buffer only, buffer with 50 mM Ca++ and buffer with 50 mM EGTA (as labeled in the pictures on the left), taken under visible light. Each petri dish was elevated using coins. The bottom panel shows the pictures taken under UV light. Only the hydrogel sample supplemented with Ca++ stayed intact at the end of the experiment.

CONCLUSION In summary, this work focused on exploiting the allosteric regulation of the β-roll domain for dual functionality. The DLeu β-roll peptides provide hydrophobic faces in the folded state, amenable to oligomerization and the 406 β-roll peptides are capable of capturing the lysozyme target. A stimulus-responsive, smart hydrogel platform was built by combining the two β-roll mutants in the same fusion protein construct. The resulting biomaterial presented in this work demonstrates the combined allosteric regulation of the viscoelastic properties and lysozyme capturing ability of the DLeu β-roll mutant and the 406 β-roll mutant, respectively. Both the self-assembly and the target entrapment within the hydrogel matrix can be controlled by adjusting the calcium concentrations. This


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hydrogel platform can be, for instance, utilized for localized cell-lysis applications, where both network formation and cell death can be manipulated. In addition, the concatemer of 406 β-roll in the hydrogel construct can be easily replaced with other proteins exhibiting different functionalities or different β-roll mutants demonstrating affinities towards other targets for tunable self-assembly and controlled release experiments.

ASSOCIATED CONTENT Supporting Information Isothermal titration calorimetry (ITC) analyses of the β-roll peptides. SDS-PAGE analysis of hydrogel forming constructs. Pictures of protein samples prepared in the presence of calcium or magnesium. Reversibility of the network assembly by the calcium chelator EGTA. Pictures of the protein samples taken under UV light following the FITC-labeled lysozyme incubation. Histogram analysis of calcium and magnesium samples via ImageJ, following the incubation with FITC-labeled lysozyme. Visualization of the hydrogel samples following the release experiments. Oligonucleotide sequences for all cloning experiments. Protein primary sequences for all constructs.

AUTHOR INFORMATION Corresponding Author Scott Banta, e-mail: [email protected]. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the US National Science Foundation (1161160 and 1402656) and the Air Force Office of Scientific Research (FA9550-12-1-0112).

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TABLE OF CONTENTS FIGURE


Conditional Network Assembly and Targeted Protein Retention via

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