Synergistic Enhancement of Enzyme Performance and Resilience via

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Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction Xue-Jian Zhang, Xiao-Wei Wang, Jiaxing Sun, Chao Su, Shuguang Yang, and Wen-Bin Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00306 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Biomacromolecules

Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction Xue-Jian Zhang1,2, Xiao-Wei Wang2, Jia-Xing Sun1, Chao Su1, Shuguang Yang*,1, and Wen-Bin Zhang*,2 1

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Center for

Advanced Low-dimension Materials, College of Material Science and Engineering, Donghua University, Shanghai 201620, P. R. China. 2

Key Laboratory of Polymer Chemistry & Physics of Ministry of Education, Center for Soft

Matter Science and Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China. RECEIVED DATE (to be inserted)

CORRESPONDING AUTHOR FOOTNOTE: Tel.: + 86 10 6276 6876; Fax: + 86 10 6275 1710; Email: [email protected]; Tel.: + 86 21 6787 4080; Fax: + 86 21 6787 4077; E-mail: [email protected]

KEYWORDS: Layer-by-layer assembly, SpyTag, SpyCatcher, SnoopCatcher, SnoopTag

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ABSTRACT. Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide-protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to bio-signals, such as Ca2+, trifluoperazine, NADPH, and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multi-layer set-up exhibits a synergistic enhancement of DHFR activity with increasing number of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience.

INTRODUCTION Proteins are the essential workhorse of life, performing a wide range of biological functions. Protein immobilization on surface is critical to take advantage of their unique functions in many applications such as drug screening, biosensors, catalytic reactors, etc.1-5 Extensive works has been carried out to develop physical and chemical immobilization techniques, such as absorption, 6,7 metal chelation,8,9 biotin-avidin interaction,10,11 native chemical ligation,12,13 NHS esterification,14 as well as various “click” reactions.15 Among various immobilization techniques, the layer-by-layer (LbL) assembly is a powerful one for surface functionalization.16-18 2


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LbL assembly, as first demonstrated by Decher,19 involves alternating deposition of polymers onto surface.16-18 As a class of most common biomacromolecules, proteins have already been used in LbL assembly, mostly as one of the components via physical interactions such as electrostatic complexation,20,21 biotin-avidin binding,22 coiled-coil interactions,23 and ConA-sugar interactions.24 Based on the elaborate works done by pioneers in LbL assembly, many natural proteins, such as glucose oxidase,25 horseradish peroxidase,25 bacteriorhodopsin,26 myoglobin,27,28 heme,29 have also been included into the multilayers to bring in intriguing properties, such as enhance enzymatic activity on colloidal particles,25 improved orientation,26 and reduced overpotential in electrochemical reduction.27,28 It has also been used to fabricate protein nanotubes30 and pH-responsive protein microcapsules,31 leading to broad applications including biocatalysts with partial or even fully maintained activity,20,32-34 biosensors,22,35,36 and protein delivery vehicles.37,38 While the other components can be synthetic polyelectrolytes,20,21 dendrimers,29 and clay nanoparticles,27,39 entirely protein-based multilayers are rare. To the best of our knowledge, there are two reported examples of entirely protein-based LbL thin films: one based on the electrostatic complexation of positively and negatively supercharged, unstructured elastin-like proteins (ELPs)40 and the other based on coadsorption of the enzyme sulfite oxidase and the electron transfer protein cytochrome c via proteinprotein interactions.41 However, electrostatic complexation requires the protein to bear a significant amount of charge and these physically associated multilayers are often not as stable as the chemically linked multilayers. Since most chemical modification of proteins tend to destabilize or inactivate proteins, it is highly desirable to establish methods that could covalently assemble proteins in a layerby-layer fashion with high efficiency, that is generally applicable to most proteins, and that could well preserve, or even reinforce their native functions after immobilization. 3



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In recent years, Howarth et al42,43 developed a set of peptide-protein reactive pairs (or “molecular superglues”) that are genetically encoded, highly reactive, and fully compatible with physiological conditions. They have received a wide range of applications, including controlling protein topology,4446

mediating colloidal assembly,47 preparing enzyme scaffolds,48 making entirely protein-based

hydrogels49,50 and “living” materials.51,52 It was also demonstrated that mutually orthogonal reactive pairs, such as SpyTag-SpyCatcher and SnoopTag-SnoopCatcher pairs, could be used to synthesize linear or branched protein polymers (“polyproteams”) with optimized combination of ligands for activating cancer cell death signal.43 We have successfully demonstrated that it could also be used to build protein multi-layers on surface, which is referred to as orthogonal “Tag-Catcher” protein chemistry enabled protein multilayer construction (OPEC).53 This approach is advantageous to the oligomerization-promoted protein LbL method using only the SpyTag-SpyCatcher chemistry54 in that it could tolerate the incorporation of a folded protein (super uranyl-binding protein, SUP) and essentially yields a chemically cross-linked, entirely protein-based, and genetically encodable thin film under mild assembly conditions. Moreover, the network structure was found to enhance the performance of immobilized SUP in uranyl sequestration with increasing number of layers. However, the scope of this method has not yet been fully explored. It remains unclear how the folded structure would impact the film growth and in which ways the multi-layered network structure would influence the catalytic activity of enzymes. Herein, we report a systematic study on the fabrication of native protein multilayers using the OPEC method. Compared to the previous study,53 we demonstrated that while most folded structures make little difference in the LbL assembly, the most extended conformation is more advantageous in promoting the assembly. It was also found that physical crosslinking, such as electrostatic 4


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complexation, can be used in synergy with protein reactions to further enhance the assembly. These unexpected findings will shed light on the design of telechelic proteins for assembly. In this work, the focus on the responsive behaviors of the protein films is shifted from relatively harsh stimuli under nonbiological context to mild bio-signals under biologically compatible conditions, such as Ca2+, trifluoperazine (TFP), nicotinamide adenine dinucleotide phosphate (NADPH), and dihydrofolate (DHF). In particular, we applied the method to make silica microparticles (SPs) functionalized with DHFR multilayers on surface and found that not only was the activity retained, but also there is a synergistic enhancement of enzyme activity with increasing number of layers. The stability enhancement was manifested by the retention of >50% of original activity after lyophilization. The results shall expand the scope of bioactive protein materials beyond the previous report53 and hold promises in prolonging the shelf-life of industrially important protein catalysts.

EXPERIMENTAL SECTION DNA Construction. The constructs of AAA-4Cys, CBC and DAD were reported previously.53 All oligonucleotide primers were ordered (Invitrogen Inc.). Genes encoding SnoopCatcher, CaM, DHFR and Affibody with designed restriction sites were ordered (Invitrogen Inc.) and cloned into the expression vector pQE-80L (Qiagen Inc.). The sequences of all constructs were verified by Sanger sequencing. Protein Synthesis and Purification. Plasmids were used to transform chemically competent Escherichia coli strain BL21. A single colony was inoculated into 6 mL of LB broth containing 100 μg/mL ampicillin and incubated overnight in a shaker at 37 °C. The overnight culture was inoculated into 300 mL of LB broth containing 100 μg/mL ampicillin and grown at 37 °C with vigorous shaking 5



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until the OD600 reached 0.5. It was then induced with isopropyl-β-D-1-thiogalactopyranoside at 22 °C for expression. The cells were harvested after 12 hours and the cell pellets were re-suspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH=8.0) and lysed by ultrasonication. The cleared lysates were mixed with a 50% Ni-NTA slurry. After agitation for 1 h, it was loaded into a column and the flow-through of the lysate was reloaded into the column for 3 times. The resin was then washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH=8.0) and the proteins were eluted by elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH=8.0). After purification, the proteins were dialyzed against PBS buffer (pH=7.4). The fluorescein-labeled proteins were prepared by mixing 2 mL of CBC-DHFR or wtDHFR in ddH2O (2.0 mg/ml) with 30 μL saturated fluorescein-5-maleimide (TCI) in ddH2O at 37 oC for 2 h. The labelled proteins were purified by ultrafiltration with ddH2O in the presence of 1 mM β-mercaptoethanol on an Amicon Ultra-0.5 centrifugal filter unit for at least 3 times. Protein Characterization. The proteins were analyzed on 10% gels for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after heating at 98 °C for 10min with 5x SDS-PAGE loading buffer (250 mM Tris-HCl, 50% glycerol, 10% SDS, 250 mM β-mercaptoethanol, 0.05% bromophenol blue). Size exclusion chromatography was performed on a Superdex 200 Increase 10/300 GL column in an ÄKTA FPLC system (GE Healthcare, Inc.) using PBS (pH = 7.4) as the mobile phase at a flow rate of 0.5 mL·min-1. Functionalization of Silica Microparticles. The method for surface functionalization was carried out according to literature report.55 Silica microparticles (SP, Aladdin, 5 μm in diameter) were silanized with 3-aminopropyltriethoxysilane for 3 h at 75°C. Then, it was incubated with a solution of

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3-(maleimido)propionic acid N-hydroxysuccinimide ester (NHS-MAL) in DMF for 45 min at 25 oC to make a maleimide-functionalized surface. Protein Quantification on Silica Microparticles. In this experiment, fluorescein-labeled wtDHFR or CBC-DHFR was used in place of unlabeled wtDHFR or CBC-DHFR to make single or multi-layered films on silica microparticles. The fluorescence at 517 nm of the resulting SP-(DHFR)n (where n is the number of bilayers) was measured by excitation at 496 nm. Standard curves of fluorescein-5-maleimide labeled wtDHFR and CBC-DHFR were made to calculate the amounts of wtDHFR and CBC-DHFR on silica microparticles. Lyophilization of DHFR Samples. The series of DHFR samples were buffer-exchanged to 50 mM NH4HCO3 buffer before freeze-drying. After two days of lyophilization, all samples were redissolved in PBS buffer with 1 mM TCEP and intensively agitated. For samples of wtDHFR and CBCDHFR, the solution were filtered through a membrane with pore size of 0.22 μm before the concentration was determined. For SP-(DHFR)5, the net weight was measured before re-suspension in PBS buffer. DHFR Activity Assay. The procedure was carried out according to previous reports.56,57 Briefly, the DHFR samples in stock solution (0.5x PBS, 1 mM TCEP) were diluted with KHP buffer (50 mM potassium phosphate containing 80.2% K2HPO4 and 19.8% KH2PO4, 5 mM β-mercaptoethanol, pH=7.5). Then, the diluted samples were mixed with 100 μM NADPH (Sigma) and 100 μM dihydrofolic acid (Sigma). Finally, DHFR samples was added into a 96-optical plate and the absorbance at 340 nm was immediately measured in kinetic mode at room temperature using EnSpire multimode plate reader (PerkinElmer Inc.). The concentration of wtDHFR or CBC-DHFR was measured by NanoPhotometerTM P-class (IMPLEMEN) with a 10 mm cuvette. A 200 μL KHP buffer 7



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was set as blank and subtracted from all data to obtain ΔOD340. NADPH standard curves was plotted to calculate the amounts of oxidized NADPH during the reaction. Linear range of all plots were used for calculation. All results were repeated in triplicate.

Figure 1. (A) Layer-by-layer assembly using orthogonal “Tag-Catcher” reactions using telechelic proteins containing folded protein domains. Surface display of functional proteins, such as GFP, can also be effected using SpyTag-functionalized GFP. (B) Gene constructs and telechelic proteins used in the current work. The folded proteins are affibody (PDB: 2KZI), DHFR (PDB: 4NX6), calmodulin in the free unbound state (CaM-1, PDB: 1CFC), in Ca2+-bound state (CaM-2, PDB: 1CLL), and in calcium- and TFP-bound state (CaM-3, PDB: 1CTR). Nonstructured ELP serves as a control. The molecular weights are shown in parenthesis. The distance between N- and C-terminus (L) is also shown. 8


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RESULTS AND DISCUSSION Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction. In line with previous report,53 two mutually complementary telechelic protein cassettes, namely CBC-X and DAD, were designed based on elastin-like protein (ELP), a modular folded domain (X), the SpyTag(A)/SpyCatcher(B) and SnoopTag(C)/SnoopCatcher(D) reactive pairs (Figure 1). Since both “Tag-Catcher” reactions require proper orientation for efficient reaction, the presence of an existing folded structure may impose considerable spatial constraint on their reconstitution. To reduce such influence and facilitate film growth, an unstructured protein, ELP, was incorporated. Folded proteins (X), such as calmodulin, affibody, and dihydrofolate reductase (DHFR), were then included in the CBC construct between the SnoopTag at N-terminus and SpyCatcher in the middle to impart function (CBC-X). To test the scope of folded proteins that can be incorporated and illustrate the effects of folded protein structures on film growth, two series of folded proteins were chosen: one based on the same protein, calmodulin (CaM), but with different conformations58-60 and the other based on proteins with distinct folded structure and sizes, namely CaM, affibody,43,61 and dihydrofolate reductase (DHFR).57,62 CaM is a dynamic protein (16.7 kDa) whose conformation can change from a relatively looselypacked structure in the free, unbound state, to extended Ca2+-bound state, and further to collapsed ligand-bound state.58-60 It has been utilized to develop dynamic hydrogels whose macroscopic motion can be controlled by changes in molecular conformation. Affibody is small protein (8.0 kDa) consisted of helix buddles with N- and C- terminus located on different sides and pointing to opposite directions. DHFR is a medium-sized protein (18.0 kDa) with proximate N- and C-terminus in space. It is 9



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anticipated that factors like folded structure, molecular weight, distance between N- and C- terminus, assembly conditions would all impact the film growth in different ways. Molecular cloning of the genes, protein expression in E. coli BL21 strain and the purification protocol are described in Experimental Section and supporting information (see Figure S1 for sequences and S2-4 for characterizations). The folded structures were confirmed by CD spectrometry and the reactivity was confirmed by mixing telechelic proteins followed by SDS-PAGE analysis. The CD spectra of CBC-CaM, CBC-Affibody, and CBC-DHFR (Figure S2) are characteristic of the αhelix signals whereas CBC-ELP is largely random coil-like.63,64 The enzymatic activity of CBC-DHFR was tested to ensure that DHFR in the fusion protein is functional. To perform the assembly, CBCCaM stock solutions was prepared in MES buffer (pH=6.0) (1 mg/mL) because the TFP ligand is only dissolved in this buffer.58 The states of CaM was controlled by adding additives, namely, 100 mM EDTA for the free state CBC-CaM-1, 10 mM Ca2+ for the Ca2+-bound state (CBC-CaM-2), and 10 mM Ca2+ and 5 mM TFP for the TFP-bound state (CBC-CaM-3), respectively. For comparison, all the assembly was also performed in MES buffer (pH=6.0). The assembly processes were monitored in situ by Quartz Crystal Microbalance with Dissipation (QCM-D) (Figure S5). The prelayer was prepared by attaching an ELP functionalized with SpyTag along the chain and four cysteine residues at the N-terminus (AAA-4Cys). The attachment of the prelayer ELP thus relies on the collective interaction between the thiol groups and the gold surface forming the Au-S bond for anchoring. Then, CBC-X and DAD was alternately deposited on surface in between which an extensive wash with MES buffer was performed to remove unreacted or nonspecifically bound proteins. Progressive decrease in frequency (∆f/n) and steady increase in dissipation (∆D) were observed in all cases (Figures 2a, S5), suggesting a robust film growth. Plot of 10


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∆f/n versus the number of layers (N) presents a linear growth profile (Figure 2b), which is not affected by the incorporation of folded domains. Nevertheless, the slope does change. A steeper slope suggests that more proteins (in terms of mass) are deposited each time. Thus, we normalized the frequency data by the differences in molecular weights of CBC-X relative to CBC-ELP (the ratio R = MCBC-X/MCBCELP).

The normalized frequency would better reflect the efficiency of interlayer reactions. It seems that

CBC-ELP, CBC-Affibody, CBC-DHFR, and CBC-CaM-1 possess essentially the same slope, suggesting that the approach is robust and could tolerate the incorporation of various ordered structures in most cases. This is in good agreement with our expectation from design that ELP shall minimize the influence of folded structure on reaction to sustain a steady and robust film growth. However, the growth curve of CBC-CaM-2 and CBC-CaM-3 is significantly higher than the rest samples. We speculated that this may be due to the presence of the divalent cation Ca2+ in the assembly buffer that was essential to maintain the CaM in different states. The ELP on CBC-X and DAD is highly hydrophilic with multiple glutamic acids. The Ca2+ may bind carboxylic acids via electrostatic interactions to promote the assembly. Similar effects have been reported by Caruso et al.65 using Cu2+ to assist weak polyelectrolyte multilayer formation on microspheres and subsequent film crosslinking. The synergy between physical and chemical interactions contributes to the much steeper slope in both cases. To prove the hypothesis, we performed the assembly of CBC-ELP and DAD in MES buffer containing 10 mM Ca2+. Compared to that in the absence of Ca2+, the assembly is significantly enhanced (Figure 2c). The slope gets steeper and is close to that of CBC-CaM-3. It suggests that the addition of Ca2+ indeed promotes the film growth by electrostatic interactions. We speculate that the crosslinking of the carboxylate charges on the chains with Ca2+ ions could screen the charges, minimize the electrostatic repulsion, and reduce the solution 11



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solubility. In this case, the difference between CBC-CaM-2 and CBC-CaM-3 could only be attributed to conformation. CaM-2 has a quite extended conformation with the N- and C-terminus located far from each other (33.96 Å), pointing to opposite direction. Both factors shall reduce the spatial constraint for reaction. By contrast, CaM-3 has a collapsed conformation with the N- and C-terminus in close proximity in space (12.58 Å), yet pointing to similar directions. Both factors lead to a crowded neighborhood and the steric hindrance may discourage binding and reaction.

Figure 2. (a) Representative QCM-D data showing ∆f/n (black) as a function of time for the assembly process using CBC-ELP (black), CBC-CaM (purple, red, and brown for free state, Ca2+-bound state, and TFP-bound state of CaM, respectively), and CBC-DHFR (blue), the pink arrows indicate the addition of CBC-X, the grey arrows indicate washing step and the green arrows indicate the adding of DAD; (b) plot of ∆f/n against the number of layers, showing linear growth profile for all samples. The ∆f/n is normalized by the ratio of molecular weight of each sample relative to CBC-ELP; (c) plot of ∆f/n against the number of layers, showing linear growth profile of CBC-ELP (empty square), CBCELP-2 (solid black square, assembled in the presence of 10 mM Ca2+) and CBC-CaM-2 (blue); (D, E) Responsive behavior of the (CBC-ELP/DAD)5 (d) and (CBC-CaM/DAD)5 (e) film to Ca2+ and TFP: the cyan arrows indicate the addition of 50 mM MES buffer (pH 6.0); the purple arrow indicates the addition of 10 mM Ca2+ in 50 mM MES buffer (pH 6.0), the red arrow indicates the addition of 5 mM 12


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TFP and 10 mM Ca2+ in 50 mM MES buffer (pH 6.0); the pink arrow indicate the adding of 50 mM Tris buffer (pH 8.0), and the brown arrow indicates the addition of 100 mM EDTA in Tris buffer (pH 8.0); (f) the (CBC-DHFR/DAD)5 film retains the substrate binding capability and catalytic activity of DHFR (the red arrow indicates the addition of KHP buffer and the brown arrow indicates the addition of 10 μM NADPH and DHF in KHP buffer). Bio-responsive and Bio-active Behaviors of the Protein Film. Successful incorporation of folded proteins will impart the film stimuli-responsive properties and unique biologically enabled functions. We first examined the film’s responsive behaviours to various bio-signals, such as Ca2+ and TFP by QCM-D. It was observed that the addition of 10 mM Ca2+ (MES, pH 6.0) leads to increase in frequency and decrease in dissipation for both (CBC-ELP/DAD)5 film and (CBC-CaM/DAD)5 film. This effect is due to the ion exchange process and the replacement of sodium ion upon binding of the divalent Ca2+ with carboxylic acids on the ELP side chains, which is consistent with previous report.53 It could be removed during extensive wash with MES buffer in (CBC-ELP/DAD) film (Figure 2d), but not in (CBC-CaM/DAD) film (Figure 2e), since CaM would bind with Ca2+ to become the CaM2 state. When the solution contains Ca2+ and TFP is added, the initial huge decrement in frequency and increment in dissipation obviously shows the binding of TFP in both cases, which adds to the mass of the film. Upon rinsing, there is a rise in frequency and reduce in dissipation with removal of nonspecifically bound TFP. The control film of (CBC-ELP/DAD)5 returned to the initial level, whereas the film of (CBC-CaM/DAD)5 stabilized at a much lower frequency consistent with the collapsed conformation of the TFP-bound CaM-3 state. The EDTA may be used to chelate the Ca2+ and reverse CaM to the free, unbound state. It was noted that EDTA was prepared in 50 mM Tris buffer (pH=8.0). Considering that pH may affect the film, we used 50 mM Tris buffer (pH=8.0) to equilibrate the film before further experiments. The increase in ∆f could be an effect of pH and/or electrolyte exchange. 13



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After the addition of EDTA (100 mM in 50 mM Tris, pH 8.0), there is a rapid increase in ∆f and decrease in ∆D for (CBC-ELP/DAD)5, but the opposite for (CBC-CaM/DAD)5. We inferred that for (CBC-ELP/DAD)5 film, it may be due to nonspecific interaction between EDTA and the protein film; for (CBC-CaM/DAD)5, the EDTA strips the Ca2+ off to form the complex accompanied by conformational transition from CaM-3 state to CaM-1 state with the loss of TFP. The complex and TFP may be trapped locally in the film, leading to apparently decreased frequency. Overall, the change is much more significant for the (CBC-CaM/DAD)5 film than that for the (CBC-ELP/DAD)5 film, which is a result of the specific interactions between CaM domain with Ca2+ and the TFP ligand. After rinsing with MES buffer again, the ∆f and ∆D of both films recover to their original states. Although the reductive activity of DHFR cannot be directly monitored by QCM-D, the binding and releasing of substrates, such as NADPH and DHF, can be tracked. The (CBC-ELP/DAD)5 film was subjected to 10 μM NADPH and DHF in potassium hydrogen phosphate (KHP) buffer. There is a slightly increase in frequency, but it quickly returned to the original value even before rinsing (Figure S6), suggesting that there is no specific interaction between the DHFR film and the two molecules (NADPH and DHF). By contrast, under similar conditions, the film of (CBC-DHFR/DAD)5 exhibits an abrupt decrease in ∆f and increase in ∆D (Figure 2f), which may correlate with the DHFR binding of NADPH and DHF. As time goes by, both the ∆f and ∆D recover a little and stabilize at certain value, which suggests that the DHFR enzymes may have reached the maximum catalytic turnover and are mostly saturated. After rinsing, the curve fully recovers to its original value, which clearly proves that the DHFR in the film can bind the substrates.

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Figure 3. (a) Silica microparticles can be functionalized with multi-layers of DHFR on surface, which retains the full catalytic capability. Surface functionalization with GFP can also be effected as shown by the confocal micrograph (scale bar: 10 μm); (b) the amount of CBC-DHFR on SP as a function of number of bilayers; (c) enzymatic activity of DHFR with increasing number of bilayers. C: control of CBC-DHFR in solution; (d) effects of lyophilization on enzymatic activity of DHFR samples. Immobilization of DHFR on Silica Particles Enhances Enzyme Activity and Resilience to Lyophilization. Enzyme immobilization on solid substrates is critical for using their catalytic activity in industrial applications.5 To translate the technique into protein materials, we planned to modify silica particles (Aladdin, diameter ~5 μm) with DHFR using this method. The maleimide functionality was first introduced to the surface of the silica particles.55 Then CBC-DHFR and DAD were alternately deposited on the surface to prepare DHFR-functionalized silica microparticle (SP-(DHFR)n where n is the number of bilayers). Notably, functionalization with another protein is possible at any step. For example, functionalization with a telechelic green fluorescent protein (GFP-AD) can be performed after CBC-DHFR to allow direct visualization of the film (Figure 3a, S7). Indeed, under confocal 15



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microscopy, these particles were all green fluorescent, confirming the successful coating of functional proteins on surface. To quantify the proteins attached during each step, we labelled the CBC-DHFR with fluoresceinmaleimide and used it in the assembly. The amounts of immobilized protein during each bilayer construction were determined by comparing the fluorescence to the standard curve (Figure S8). It was found that the amounts of CBC-DHFR increased steadily with increasing number of bilayers (Figure 3b), consistent with the linear growth mode revealed by QCM-D. We also determined the activity during each bilayer construction using the kinetic mode (Figure S9). The results are shown in Figure 3c and compared to that of the CBC-DHFR in solution. It was found that the immobilization of DHFR initially led to compromised activity as compared to that in solution. This is common for most proteins and is rationalized through the confinement effect on surface and the structure network which make them more rigid and less accessible to the reactants in solution. Nevertheless, a steady increase in activity was observed with increasing number of bilayers. For thin films of 4 layer or more, the DHFR enzymes already have an apparently higher activity than their solution states. Notably, the SP-(DHFR)5 shows an activity that is 76% better than the CBC-DHFR in solution. The enhancement may be attributed to the synergistic effects among adjacent layers and neighboring proteins. The top layers are more exposed in solution and are more mobile and more efficient in capturing NADPH and DHF into the thin film. Moreover, the bottom layers are not “buried”, but at least equally active. Once the NADPH and DHF get into the film, they are exposed to many DHFR units and are less likely to escape before reaction, leading to an apparently much higher enzyme activity. The multivalent effect is reminiscent of our recent finding that protein catenane containing DHFR exhibits higher activity than the corresponding monomer in solution.62 16


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Serendipitously, we found that this method also made the protein more resilient to handling. Enzymes are known to be sensitive to extensive manipulations, such as freeze-drying or multiple cycles of freeze-and-thaw. For example, the wild-type DHFR (wt-DHFR) would lose ~96% of its activity upon freeze-drying and re-dissolution in PBS buffer (pH=7.4, 1 mM TCEP). Under similar conditions, the fusion protein CBC-DHFR loses ~99% of its original value. By contrast, the thin film of (CBC-DHFR/DAD)5 on silica particle, or SP-(DHFR)5, retains up to 50% of its original activity (Figure 3d). We speculated that during freeze-drying, DHFR may unfold and aggregate due to hydrophobic interactions and random disulfide bond formation. They are trapped in this metastable state in solution. However, the confinement of DHFR on surface within the network of highly hydrophilic ELP segments minimizes the irreversible aggregation, reinforcing the resilience of DHFR. The multi-layered set-up with the “protective” ELP matrix thus holds great promise in extending the shelf-life of functional proteins.

CONCLUSION In summary, we have shown that orthogonal peptide-protein chemistry enables a very robust layer-by-layer process that facilitates the incorporation of various folded proteins. Not only are the folded structures completely preserved, but also their functions are reinforced. As dictated by the unique functions of the immobilized protein, the film could exhibit stimuli-responsive behaviors to different biological signals and catalyze chemical transformations. Notably, with increasing number of layers, the amounts of immobilized protein are increased and their enzymatic activity are reinforced, outperforming their solution counterparts. Moreover, the protein multilayer also confers considerable resilience on DHFR to lyophilization, which shall help extend the shelf-life and facilitate the storage 17



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and shipment of functional proteins. The study expands the scope of protein-based materials and offers an approach to integrate their unique biological functions onto synthetic materials with potential synergistic enhancement of activity and resilience.

ACKNOWLEDGEMENTS We are grateful for the financial support from the National Natural Science Foundation of China (Grants 21474003, 91427304), the 863 Program (2015AA020941), and “1000 Plan (Youth)”.

SUPPORTING INFORMATION Amino acid sequences, SDS-PAGE images, CD spectroscopy, other characterization data and additional experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

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For Table of Contents Use Only Title: Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal PeptideProtein Chemistry Enabled Multilayer Construction Authors: Xue-Jian Zhang, Xiao-Wei Wang, Jia-Xing Sun, Chao Su, Shuguang Yang*, and Wen-Bin Zhang*

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Synergistic Enhancement of Enzyme Performance and Resilience via

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