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A Versatile and Robust Approach to Stimuli-Responsive Protein Multilayers with Biologically Enabled Unique Functions Xue-Jian Zhang, Xiao-Wei Wang, Xiao-Di Da, Yanlin Shi, Chunli Liu, Fei Sun, Shuguang Yang, and Wen-Bin Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00190 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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Biomacromolecules

A Versatile and Robust Approach to Stimuli-Responsive Protein Multilayers with Biologically Enabled Unique Functions Xue-Jian Zhang1,2, Xiao-Wei Wang1, Xiao-Di Da1, Yanlin Shi3, Chunli Liu3, Fei Sun4, Shuguang Yang*,2, Wen-Bin Zhang*,1 1

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 2

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 3

Beijing National Laboratory for Molecular Sciences, Fundamental Science Laboratory on

Radiochemistry & Radiation Chemistry, College of Chemistry & Molecular Engineering, Peking University, Beijing 100871, P.R China 4

Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, P.R. China E-mail: [email protected]; [email protected]

RECEIVED DATE (to be inserted)

CORRESPONDING AUTHOR FOOTNOTE: Tel.: + 86 10 6276 6876; Fax: + 86 10 6275 1710; E-mail: [email protected]; Tel.: + 86 21 67874080; Fax: + 86 21 67874077; E-mail: [email protected]

KEYWORDS: SpyTag, SpyCatcher, orthogonal reactions, layer-by-layer assembly, SUP, uranium mining 1 ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT. Protein-based materials call for innovative processing techniques to integrate their unique biologically enabled functions with other materials of complementary features. Herein, we report the covalent protein layer-by-layer assembly via orthogonal “Tag-Catcher” reactions as a facile and robust approach to make entirely protein-based multilayers on a variety of substrates. Programmed assembly of native telechelic proteins not only endows the materials valuable stimuli-sensitive behaviors, but also unique properties unparalleled by any synthetic counterparts. As proof of concept, super uranyl-binding protein (SUP) is immobilized on silica gel by this method with tunable capacity and enhanced capability for uranyl sequestration. Not only is the capturing performance enhanced in the multilayer setup, it also confers resilience to recycling, allowing efficient harvest of uranyl with an average of ~90% and ~60% recovery rate in over 10 cycles from water and synthetic seawater, respectively. The approach is the first entirely-protein-based multilayers covalently assembled by the layer-by-layer method. It provides a platform for immobilizing proteins with synergistic enhancement of function and resilience and expands the scope and capability of genetically encoded protein-based materials.

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INTRODUCTION Surface/interface engineering with biomacromolecules is critical in improving materials’ biocompatibility and bioactivity.1-6 As the workhorse of life, proteins are a unique class of biomaterials for a wide range of applications.7,8 It takes creative ways to process them into functional forms like gels and films without compromising their native structures and functions to unleash their full potential as materials.9-11 Layer-by-layer (LbL) assembly, firstly demonstrated by Decher,12 is an well-established technique for surface functionalization involving alternate deposition of two (or more) building blocks with mutually complementary interactions.13-16 Due to their vulnerable structures and relatively high costs, proteins are much less used in LbL assembly and are often used only as part of the assembly components to complex with synthetic polyelectrolytes.17-23 Noncovalent interactions, such as coiled-coil interactions, biotin-avidin complexation, and ConA-sugar interactions, have been used to assemble protein-containing multilayers.24,25 An entirely protein-based LbL system was reported by Kolbe et al.26 based on the electrostatic complexation of positively and negatively supercharged elastin-like proteins (ELPs), which is unstructured and similar to synthetic polyelectrolytes.27 These physically associated films are usually not as stable and robust as chemically cross-linked films. Nonetheless, it remains a challenge to conveniently construct the multilayer with covalently cross-linked native proteins. It is also desired that such technique could help boost proteins’ stability and/or activity.28-30 The emergence of the “molecular superglues” has brought new opportunities to the field.31,32 They were developed by splitting natural isopeptide-forming domains (e.g. CnaB2) into a peptide tag and the cognate protein partner, such as the SpyTag/SpyCatcher33,34 and SnoopTag/SnoopCatcher reactive pairs.35 They reconstitute to restore the autocatalytic activity and form the isopeptide bond with high reactivity, rapid kinetics, and high selectivity under physiological conditions.36 To date, they have received numerous applications from engineering protein topology,37 preparing mechanically interlocked protein architectures,38-40 making protein 3 ACS Paragon Plus Environment


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gels,37,38,41 controlling cellular location42 and membrane function,43 developing synthetic vaccines,44 to living materials.45 Notably, orthogonal reactivity can be encoded for the same reaction, either for those from distinct ancestor domains35 or from the same precursor with as few as three mutations.46 We envisioned that mutually complementary telechelic proteins from orthogonal reactive pairs could be used to modularly assemble all-protein-based films following the repetitive LbL protocol. Such orthogonal pairs have been used to make protein polymers (or polyproteams) for optimal multi-protein ligand activity.35 Herein, we report a robust, efficient, and versatile platform to fabricate covalently cross-linked, stimuli-responsive, all-protein-based multi-layers containing folded protein domains (Figure 1), which is referred to as the orthogonal “Tag-Catcher” protein chemistry enabled multilayer construction. There are several advantages of this approach: (1) the reactions occur spontaneously with no additional coupling reagents or unnatural amino acids; (2) it results in a stable, chemically crosslinked network whose properties are fully encoded on the genetic level; (3) it facilitates the modular incorporation of folded proteins with well-preserved activity. To demonstrate the utility, we identified selective uranyl mining from the ocean as a potential application, which has been listed as one of the seven chemical separation challenges to change the world.47 The uranium reserve in ocean, typically in the form of uranyl ion (UO22+) at only 3.2 ppm, has a surprisingly high total amount that is ~1000 times more than that in land.48 However, synthetic polymers have only unsatisfactory performances to date in terms of their binding affinity and selectivity.49 Recently, super uranyl-binding proteins (SUPs) have been engineered to possess femtomolar affinity and >10,000-fold selectivity over other metal ions.50,51 It has been used in SUP-containing hydrogels for uranyl sequestration.52,53 It would be desirable to integrate this unique property on mechanically robust synthetic materials like silica gels. We demonstrate the utility of this technique in modifying silica gel with surface coated SUP multi-layers (SG-(SUP)n) and show that it can be used for effective uranyl sequestration and endure multiple recycling processes 4 ACS Paragon Plus Environment

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Figure 1. Building protein multilayers using layer-by-layer assembly via orthogonal “Tag-Catcher” reactions.

EXPERIMENTAL SECTION DNA Construction: All oligonucleotide primers were ordered from Invitrogen. Genes encoding SnoopCatcher, SUP with designed restriction sites were assembled by overlapping PCR and cloned into the bacterial expression vector pQE-80L (Qiagen Inc.) by standard restriction digestion and ligation protocols. The full sequences were confirmed by direct sequencing. Protein Synthesis and Purification: Plasmids were used to transform chemically competent E. coli strain BL21 for expression. Proteins were purified according to standard protocols of metal affinity chromatography using lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH=8.0), wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH =8.0) and finally, the proteins were eluted by elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH=8.0) (see SI). After purification, the proteins were



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dialyzed against PBS buffer (pH = 7.4) Protein Characterization: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on10% gels to analyze the proteins. Samples were mixed with 5x SDS-PAGE loading buffer (250 mM Tris-HCl, 50% glycerol, 10% SDS, 250 mM β-mercaptoethanol, 0.05% bromophenol blue) and heated at 98 °C for 10 min. 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. QCM-D Measurement: The protein samples were dissolved in PBS (pH=7.4). The growth of the films was monitored in situ by a home-made quartz crystal microbalance and a flow chamber. For all measurements, quartz crystal chips with gold coated surface with a fundamental frequency of 5 MHz were washed by ethanol and pure water in high speed, alternately for 3 times at least, and finally dried by nitrogen stream. All measurements were made with the crystals in flow cells where only the upper side of the crystals was in contact with the solution, and the system is under injection with a flow rate of 50µL/min. Before the experiment, all solutions were filtered by a membrane with pore size of 0.22 µm and further centrifuged at 3500 rpm for 30 minutes to remove bubbles. For the film buildup, the assembly solutions are flowed through the crystal for 30 min, followed by washing with PBS buffer for 20 min. Protein Labeling: Fluorescein-5-Maleimide (TCI) was used to make fluorescently labeled proteins including wt-SUP and CBC-SUP. The protein (CBC-SUP or SUP in ddH2O, 2 ml, 2 mg/mL) was mixed with 30 µl saturated Fluorescein-5-Maleimide solution at 37 oC for more than 2 hours. Then, the product was purified by centrifugation with ddH2O in the presence of 1 mM β-mercaptoethanol for more than 3 times. Functionalization of Silica Gel Surfaces: The method for surface functionalization was carried out similar to literature report.7 The silica gel (SG) was silanized with 3-aminopropyltriethoxysilane for 3 hours at 6 ACS Paragon Plus Environment

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75°C. Then, a solution of 3-(maleimido)propionic acid N-hydroxysuccinimide ester (NHS-MAL) in DMF was used to prepare maleimide-functionalized surface by incubating for 45 min at 25 oC. Quantification of Immobilized Protein on Silica Gel: Fluorescein-5-maleimide labeled CBC-SUP or SUP was used to replace CBC-SUP or SUP to make single or multi-layered film on SG. The fluorescence at 517 nm of the resulting SG-(SUP)n (where n is the number of bilayers) was measured by excited at 496 nm. Standard curves of fluorescein-5-maleimide labeled CBC-SUP or SUP were also made to quantify the amounts of CBC-SUP or SUP protein on silica gel. Assembly of SG-(CBC)5: SG-MAL was first functionalized with AAA-4Cys (1 mg/ml, PBS buffer) to form the prelayer by incubating at 25 oC for 20 min. Then, the material was alternately soaked in a solution of CBC-SUP or DAD (1 mg/ml, PBS buffer) for 15 min each. After each procedure, the SG was harvested by centrifuged at 5000 rpm for 1 min and the solution was decanted. In between the two steps, there is a rinsing step with PBS buffer. Uranyl Sequestering Experiments: For experiments assessing the uranyl capturing ratio, the uranyl is used in excess over SUP. The UO2(NO3)2 (Spectrum Chemical, ACS Grade Reagent) (500 µL, 10 µM in ddH2O) was added in to 1.5 ml Eppendorf tubes containing surface-modified silica gel (20 mg) and agitated for 10 min. Then, the tubes were centrifuged to remove the UO22+ solution and further washed by ddH2O (1 mL each, 3 times). Finally, the SG was soaked in 200 µL 0.02 M NH4CO3 solution for 10 min. After centrifugation at 5000 rpm, the supernatant was decanted and the concentration of UO22+ recovered from silica gel was determined using Arsenazo III method.54 As for the experiment on the effect of SUP excess on the UO22+ recovery rate, the UO22+ added is kept constant (500µL, 1µM) and the amount of protein added is changed by adding different amount of SG-(SUP)5. Uranyl Recovery Experiments: For recycling experiments, UO22+ (1 mM) was diluted into 2L volume 7 ACS Paragon Plus Environment


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ultrapure water (18.2 MΩ) to make a 10 nM final UO22+concentration. The solution was transferred into the column loaded with 0.5 ml SG-(SUP)5 in portions (200 mL*5). After that, it was washed by passing ultrapure water through the column (10 mL). The NH4CO3 solution (300 µL 0.02 M) was added into the column and soaked with SG-(SUP)5 for more than 10 min before it was eluted. The process was repeated for 3 times. The uranyl concentration was then determined following the literature procedure using Arsenazo III method.54 The Arsenazo III (80 µM, 50 ml) containing 0.1 M HCl was titrated with an equal volume of uranyl solutions ranging from 0 to 10 µM and the absorbance at 652 nm was monitored to give the standard curve. For uranyl recovery experiment in synthetic water, the artificial sea water was prepared according to literature55 with UO22+ added at a final concentration of 10 nM. The recovery experiments were conducted in a way similar to the above experiments in ddH2O. The only difference is that the UO22+ was eluted by 6 mL of NH4CO3 solution (0.02 M). The concentration of UO22+ was measured by ICP-MS (NexlON 350X, PerkinElmer).

RESULTS AND DISCUSSION Orthogonal “Tag-Catcher” Protein Chemistry Enabled Protein Covalent Layer-by-layer Assembly. In principle, a steady and continual growth of an LbL film requires both efficient complexation and consistent regeneration of sufficient surface reactive sites. We have previously attempted to build protein multi-layers using only the SpyTag-SpyCatcher chemistry. Two telechelic elastin-like proteins (ELPs), AA and BB where A stands for SpyTag and B for SpyCatcher,37 were used. The film growth ceased after ~4 layers due to gradually diminished surface reactive groups (Figure S1). Orthogonal chemistry is thus a must to sustain the film growth. Two telechelic ELPs bearing complementary reactive sequences, namely CBC and DAD (Figure S2) where C stands for SnoopTag, and D for SnoopCatcher, were designed. The rationale to use ELP is two-fold: (1) ELP is disordered in solution and the flexible chain alleviates the stringent demand on the spatial 8 ACS Paragon Plus Environment

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relationship for efficient reaction; and (2) it provides a matrix for modular loading of any protein of interest into the film. An ELP containing three SpyTag and four cysteine residues at the C-terminus (AAA-4Cys) was designed as the pre-layer (Figure S3). Crosslinking occurs with each deposition, adding to the stability of the film. One of the ELP block in CBC could be replaced with a folded protein like SUP50 to give CBC-SUP (Figure S2). SUP exists as a multi-helix bundle capable of capturing uranyl with high affinity. The genes were synthesized and expressed in E. coli. BL21 strain in LB medium at 22 oC for 12 hours with IPTG induction. The proteins were purified by Ni-NTA affinity chromatography and obtained in good yields (~40-70 mg/L). The characterization by SDS-PAGE, SEC, and mass spectrometry confirmed their purity (Figure S4-S7) and the CD spectra further corroborated the existence of folded structures in CBC-SUP (Figure S5). The orthogonal reactivity was confirmed by mixing respective reactants and determining the product distribution in SDS-PAGE (Figure S5) before the assembly was performed. Quartz crystal microbalance with dissipation (QCM-D) was used to characterize the protein film and track the film growth and property changes.56,57 A sharp decrease in frequency (~20 Hz) was observed for the prelayer formation, indicating efficient attachment of the proteins (Figure S8). The f is the frequency parameter and D is the dissipation parameter measured by QCM-D. Considering that the ∆D of the film is relatively low (~2), one can assume that the prelayer is relatively rigid so that we can estimate the mass of the prelayer using the Sauerbrey equation (∆m=-∆f▪C where C = -17.7 ng/cm2 for a 5 MHz quartz crystal).56,58 The results is roughly ~350 ng/cm2, corresponding to an intermediate graft density of ~0.1 nm-2.59 Then, CBC and DAD were alternately injected into the flow cell for sequential assembly, in between which there is a rinsing step. A sharp decrease in frequency shift (∆f/n) can be seen after the injection of each telechelic protein, suggesting their effective attachment (Figure 2a). Extensive rinsing caused the ∆f/n to increase a little, indicative of the removal



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of nonspecifically associated species or entangled chains.60 The net change of ∆f/n after wash thus represents only the covalently-linked thin layer of proteins. Plotting ∆f/n against the number of layers shows a robust linear growth profile (Figure 2c).56,61 This is in sharp contrast to the jeopardized film growth using only one reaction (Figure S1). When CBC-SUP is used instead of CBC, the growth profile remains essentially unchanged as the original CBC/DAD system (Figure 2b), which demonstrates again the robustness of this method. The resulting film is named (CBC/DAD)N or (CBC-SUP/DAD)N with N being the number of bilayers. The film of (CBC/DAD)5 on gold surface was characterized by AFM (Figure S9). The surface is very smooth, suggesting a homogenous film. The thickness was measured to be ~17 nm by scratch, which is consistent with the estimation from QCM data by the Sauerbrey equation (~20 nm). The successful assembly enables transfer of the structural/functional properties of proteins into desired macroscopic properties of the materials. Stimuli-responsiveness is perhaps the most common trait for proteins. The ELP used in this work is sensitive to the changes in pH, ionic strength, temperature, and metal ions. We anticipated that the resulting film should faithfully exhibit such responsive behaviors. Stimuli-responsive Behaviors.. We first examined the film’s response to different pH values. The (CBC/DAD)5 film was sequentially equilibrated with PBS buffers at different pH values ranging from 9.5 to 3.5. The ∆f/n of (CBC/DAD)5 normalized to that at pH=9.5 was plotted against the corresponding pH values (Figure 2d, S10). It is clear that the ∆f/n and dissipation (D) first decreases upon lowering the pH. After reaching a minimum at pH ~6.5, the ∆f/n starts to rise again before it levels off at even lower pH. This is closely related to proteins’ charged states. At higher pH, the carboxylic acids are deprotonated and the electrostatic repulsion takes over, leading to the swelling of the film. At lower pH, the carboxylic acids are neutral but the amine groups are protonated, contributing again to the electrostatic repulsion and swelling. Only at pH around 10 ACS Paragon Plus Environment

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the isoelectric point (pI), the protein exists in zwitterionic form and strong electrostatic attraction between oppositely charged side groups leads the film to shrink. Compared to the theoretical pI values of CBC (3.8) and DAD (5.8), the observed minimum around pH~6.5 is reasonable since pKa values may vary due to local environments. Similar trend has been observed in pH responsive intrinsically disordered protein brushes and resilin-mimetic protein bilayers.62

Figure 2. QCM-D data showing ∆f/n (black) and ∆D (blue) as a function of time for the assembly process using CBC (a) or CBC-SUP (b) and DAD. The purple arrows show the addition of CBC or CBC-SUP; the red arrows indicate the addition of DAD; the green arrows indicate the PBS washing step. (c) Plot of ∆f/n against the number of layers, showing robust linear growth profile for both samples. The resulting film of (CBC/DAD)5 shows responsive behavior to pH (d), ionic strength (e), temperature (f), and divalent ions like Ca2+ (g) and 11 ACS Paragon Plus Environment


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UO22+ (h), whereas the film of (CBC-SUP/DAD)5 could selectively absorb UO22+ (i). The red square indicates the specific interaction between UO22+ and (CBC-SUP/DAD)5. The frequency data, ∆f/n (black), and dissipation data, ∆D (blue), are for the overtone n=3. For (g-i), the red arrows show the addition of metal ion, the blue arrows show the adding of Bis-Tris buffer, the purple arrows show the adding of Tris buffer and the orange arrow show the adding of eluent agents.

Increasing ionic strength causes a similar dual effect on the ELP surface layers (Figure 2e, S11). When the film of (CBC/DAD)5 was equilibrated with PBS buffer with increasing [NaCl] at pH = 7.5, the ∆f/n was found to increase first at lower ionic strengths (

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