B12-Dependent Protein Oligomerization Facilitates Layer-by-Layer

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Letter Cite This: ACS Macro Lett. 2018, 7, 514−518

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B12-Dependent Protein Oligomerization Facilitates Layer-by-Layer Growth of Photo/Thermal Responsive Nanofilms Jingjing Wei,†,‡ Wen-Hao Wu,† Ri Wang,§ Zhongguang Yang,§ Fei Sun,*,§ and Wen-Bin Zhang*,† †

Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, Center for Soft Matter Science and Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, People’s Republic of China ‡ College of Chemical and Environmental Engineering, Anyang Institute of Technology, Anyang, Henan 455000, People’s Republic of China § Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR China S Supporting Information *

ABSTRACT: We report the robust growth of an entirely protein-based, photo- and thermoresponsive Layer-by-Layer nanofilm using genetically encoded SpyTag/SpyCatcher chemistry. The process was facilitated by AdoB12-induced tetramerization of photoreceptor proteins. Protein cargos can be released from the film in a light-dependent manner, showing its potential for therapeutic protein delivery.

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irected assembly of protein molecules into high-order architectures has proven effective for creating bioactive composite materials.1−3 Over the past decades, layer-by-layer (LbL) assembly has matured into a versatile surface/interface engineering technology to create molecular multilayers with well-defined thickness and composition.4−6 While synthetic polymers are commonly used, there are also reports on using proteins to create LbL multilayers by noncovalent interactions such as electrostatic, coiled-coil, avidin−biotin, and lectin− sugar interactions.7−9 Yet, the ecological diversity of naturally occurring proteins remains to be fully exploited to develop functional nanofilms with intriguing properties such as photoresponsiveness. Photoresponsive proteins have lately emerged as powerful optogenetic tools for biological studies.10−12 Their potential in creating dynamic biomaterials with tailored properties is just about to unfold.13−16 UVR8-derived protein was first used as a self-assembling peptide−protein conjugate to make UVsensitive hydrogel.16 The C-terminal adenosylcobalamin (AdoB12)-binding domain of CarH protein (CarHC) tetramerizes upon binding to AdoB12 in the dark and readily dissociates into monomers accompanied by the photolysis of AdoB12 and a drastic protein conformational change upon exposure to green light (Figure 1A).17,18 This was used to make a photoresponsive protein hydrogel for controlled release of proteins/ cells by covalently polymerizing the CarHC domains.13 Wu et al. recently synthesized a reversible photoresponsive hydrogel enabling optically controlled cell migration through the conjugation of 4-arm-PEG-maleimide and Dronpa, a GFP variant that tetramerizes and disassembles in a light-dependent manner.14 Photoresponsive hydrogel based on Dronpa was also reported by Li et al.19 It would be desirable to use these motifs in making photoresponsive protein nanofilms. © XXXX American Chemical Society

Figure 1. (A) AdoB12 binding induces CarHC tetramerization in the dark. Light (522 nm) exposure disassembles tetrameric CarHC accompanied by the degradation of AdoB12 and the release of 4′,5′anhydroadenosine. CarH C tetramer, PDB code: 5C8A; CarHC monomer, PDB code: 5C8F. (B) Loop formation and reactive group quenching impede the assembly of monomeric ACA and BCB in the absence of AdoB12. (C) CarHC tetramerization induced by AdoB12 leads to the formation of multivalent (8) building blocks, (ACA)4 and (BCB)4, which promotes immobilization and faciliates the LbL assembly.

Genetically encoded SpyTag/SpyCatcher chemistry, a peptide/protein pair that can spontaneously form a stable isopeptide bond between the side chains of Lys and Asp, has Received: February 17, 2018 Accepted: April 3, 2018

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DOI: 10.1021/acsmacrolett.8b00147 ACS Macro Lett. 2018, 7, 514−518

Letter

ACS Macro Letters ushered in a paradigm shift for designing protein materials.20−24 In principle, its marked efficiency and selectivity under physiological conditions would make it ideal for making chemically cross-linked, all-protein-based multilayers. However, LbL assembly using a single type of ligation chemistry often suffers from retarded film growth, owing to reduced reactive sites with loop formation and inaccessible surface during the LbL process (Figure 1B). Natural evolution has enabled biological systems to figure out a way to achieve multivalence and cooperativity by simply clustering together multiple reactive sites via oligomerization.25 We envisioned that this strategy may also be used to circumvent the reactive site quenching by defining the spatial arrangement of multivalent reactive groups (Figure 1C). In this communication, we show that B12-dependent CarHC tetramerization can facilitate a robust LbL growth of multistimuli-responsive protein multilayers on the Au surface using only SpyTag/SpyCatcher chemistry. Two complementary telechelic proteins were designed based on thermally responsive elastin-like protein (ELP) and photoresponsive CarHC protein: SpyTag-ELP-CarHC-ELPSpyTag (ACA) and SpyCatcher-ELP-CarHC-ELP-SpyCatcher (BCB) (Figure S1).13 A prelayer protein, SpyTag-ELP-CarHCELP-SpyTag-4Cys (ACA-4Cys), was designed with four cysteine residues on the C-terminus (Figure S2). It was meant to form a prelayer on the gold surface via Au−S bond and support further alternate assembly of ACA and BCB into a multilayer structure through SpyTag/SpyCatcher chemistry (Figure 1). The elastin-like polypeptide (ELP) domain composed of repeating pentapeptides, (VPGXG)15, where X is either Val or Glu in a 4:1 ratio, was chosen for its high expression yield in Escherichia coli as well as marked phase transition behavior in response to stimuli-like temperature and ionic strength. Incorporation of ELP and CarHC into a protein multilayer may enable the direct transfer of their stimuliresponsiveness at the molecular level to material properties at a macroscopic scale for applications like photocontrolled delivery. A protein cargo functionalized with CarHC domain, mCherry-CarHC, was also designed (Figure S3) as the model compound to test the photoreleasing behaviors of the resulting film. The proteins were produced in decent yields via expression in E. coli BL21 strain and purified by Ni-NTA chromatography. According to the size-exclusion chromatography (SEC) analysis, both ACA and BCB proteins were monomeric in the absence of AdoB12 and tetramerized upon addition of AdoB12 in the dark as expected (Figure 2A,B). Au surface was used to support the assembly of protein nanofilms. It was first treated with the PBS solution containing ACA-4Cys followed by extensive wash with PBS. The prelayer formation was confirmed by the decrease in frequency shift according to the Quartz Crystal Microbalance with Dissipation (QCM-D) analysis (Figure 2C,E). It should be noted that cysteine residues are essential for efficient prelayer formation. A control experiment was performed with the ACA protein lacking cysteine residues (Figure S4). There is very little nonspecific adsorption to the surface, as reflected by the change as small as ∼1 Hz upon addition of ACA. In addition, these nonspecifically associated proteins were readily removed by washing. Alternate addition of BCB and ACA on the ACA-4Cysdecorated Au surface was supposed to result in the stepwise growth of a protein multilayer as a result of the covalent conjugation by SpyTag/SpyCatcher. However, the LbL

Figure 2. (A, B) SEC analysis of oligomeric states of ACA and BCB in the absence and presence of AdoB12. (C, E) QCM-D data showing growth modes of LbL protein films in the absence (C) and presence (E) of AdoB12. (D, F) LbL assembly profiles of monomers, ACA + BCB (D), and tetramers, (ACA)4 + (BCB)4 (E). The frequency data, ΔFn (black) and ΔD (green) are for the overtone n = 3. In (D) and (F), the protein mass is the hydrated mass of the protein layer.

assembly in the absence of AdoB12 could not persist and ceased at a film as thin as 6 layers (Figure 2C,D). On the contrary, the addition of AdoB12 significantly improved the LbL assembly efficiency of BCB and ACA in the dark and led to the growth of a much thicker protein multilayer (≥20; Figure 2E). It exhibited a robust linear growth mode in sharp contrast to the one without AdoB12 (Figure 2F). The stark difference may arise from the distinct oligomeric states of ACA and BCB caused by AdoB12; the higher LbL assembly efficiency in the presence of AdoB12 may reflect the combined contribution from the cooperative effects and structural constrains of tetrameric folded proteins during the immobilization reaction (Figure 1C). The tetramerization of ACA and BCB significantly increased their valency from 2 to 8, which not only minimized the possibility of loop formation but also increased the immobilization efficiency as the film grew (Figure 1). Previous studies showed that the reconstitution efficiency of SpyTag and SpyCatcher in PBS is ∼80% within 15 min.20 The LbL assembly efficiency of monomeric ACA and BCB in the absence of AdoB12 largely depends on that of SpyTag/ SpyCatcher chemistry. Assuming the probability for ACA4Cys to immobilize onto Au surface (layer 1) is 1, the subsequent immobilization probability for each monomeric BCB molecule (layer 2) should approximate the SpyTag/ SpyCatcher reconstitution efficiency (0.8) and the immobilization probability for a molecule on layer 6 would be drastically reduced to 0.3277, given p = 0.8N−1 on layer N. By contrast, the immobilization probability for each tetrameric (BCB) 4 molecule in the presence of AdoB12 is 1−(1−0.8)4 = 0.9984, 0.9920, 0.9700, and (0.9984)N−1 on layer 2, 6, 20, and N, respectively, substantially higher than that of its monomeric counterpart (p = 0.8, 0.3277, 0.0144, and 0.8N−1). Although the 515

DOI: 10.1021/acsmacrolett.8b00147 ACS Macro Lett. 2018, 7, 514−518

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ACS Macro Letters

of divalent metal ions by EDTA restored frequency-shifts, showing that the direct metal binding of the protein film was responsible for the observed responsiveness. To ensure that the changes are not just due to changes in buffer composition, a control experiment was performed on bare Au surface that does not have ELP (Figure S6). Both the magnitude and the direction of changes are very different, again confirming the roles of ELP in the ion responsiveness. Building chemically cross-linked, entirely protein-based multilayer provides a platform to take advantage of the versatile and unique functions of protein molecules. The AdoB12 induces CarHC tetramerization under dark conditions, while light exposure cleaves the axial Co−C bond of AdoB12, triggers a protein conformational change and disassembly of tetrameric CarHC into monomers. We envisioned that the CarHC protein film could serve as a delivery device for the loading and unloading of protein cargos in a light-dependent manner (Figure 4A). Using mCherry-CarHC as a model cargo, we synthesized a protein multilayer (20 layers) laden with mCherry-CarHC under dark conditions (Figure 4B,C). The QCM-D measurement revealed a steady linear growth mode despite the presence of a significant amount of mCherryCarHC. At a stoichiometric ratio of 1/3 (for mCherry-CarHC/ ACA or mCherry-CarHC/BCB), they can efficiently form the

coupling efficiency at the interface may be expected to be even lower than 0.8, the stark contrast between the two scenarios is unchanged. This inducible protein oligomerization may represent a general strategy for improving the efficiency of creating multilayer structures on solid substrates. ELPs are thermally responsive and undergo a unique phase transition around the temperature known as lower critical solution temperature (LCST). The proteins are hydrophilic at low temperature (LCST). The ELP in ACA and BCB is very hydrophilic.26 To examine the influence of temperature on the protein film comprising (ACA)4 and (BCB)4, QCM-D was used to measure its frequency-shift under stepwise heating (25 → 60 °C) and cooling (60 → 25 °C; Figure 3A). Increased

Figure 3. (A) QCM-D data showing the influence of stepwise heating from 25 to 60 °C and cooling from 60 to 25 °C with a 5 °C interval. (B) Plot of frequency shifts resulting from heating protein multilayers with varied thickness (6 and 10 layers) from 25 to 60 °C. Responsiveness of protein films toward (C) Mg2+ and (D) Ca2+ (10 mM).

frequency-shift was observed as the temperature increased and there is a change in slope in the plot of frequency versus temperature (Figure 3B), suggesting dehydration of the film due to the hydrophobic aggregation of the ELP domains. Upon cooling, frequency-shift decreased, reflecting the rehydration of a more hydrophilic protein film at lower temperatures (Figure 3A). However, the slope does not change, indicating a big hysteresis due to the chemically cross-linked structure (Figure S5). It is also noteworthy that the thermal responsiveness of the films was independent of the thickness of protein films, as both 6-layer and 10-layer films exhibited similar transition temperatures (Figure 3B). It suggests that the thermal responsiveness of protein films is an inherent property encrypted into the protein sequence. Divalent ions such as Mg2+ and Ca2+ can lower LCST and trigger the phase transition by binding to the carboxylates of the Glu residues within ELP, a process often accompanied by loss of water molecules (dehydration) from the protein polymers.26 We investigated the effects of Mg2+ and Ca2+ on our ELP-containing protein films (10 layers in thickness) assembled in PBS buffer free of divalent ions. It turned out that the addition of 10 mM Mg2+ and Ca2+ indeed led to increased frequency-shifts, showing the dehydration of the film presumably caused by increased hydrophobicity of ELPs upon metal binding/cross-linking (Figure 3C,D). The removal

Figure 4. (A) Scheme of optically controlled release of loaded protein cargos such as m-Cherry (mC, PDB code: 2h5q) from LbL films. (B) QCM-D data showing that the incorporation of mCherry-CarHC does not impede the film growth. The frequency data, ΔFn (black) and ΔD (green) are for the overtone n = 3. (C) Linear growth mode of an LbL protein film in the absence (blue) and presence (red) of mCherryCarHC. (D) QCM-D data showing the release of mCherry from the assembled film in response to pulsed white LED light (80 W) with varied intervals (purple) or with continuous illumination (pink). Green-yellow and dark red arrows indicate light on and off, respectively. (E) Plot of the amounts of released mCherry-CarHC from the film as a function of white LED illumination time. The red dot is the starting point and the purple triangle is the end point, indicating that after about 15 min of illumination, no more protein is released. 516

DOI: 10.1021/acsmacrolett.8b00147 ACS Macro Lett. 2018, 7, 514−518

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ACS Macro Letters oligomers (Figure S7) and promote the LbL assembly (Figure S8A). Intriguingly, there appears a transition in slope with increasing number of layers (Figure 4C). Such a transition can be rationalized by the change in reactive sites on surface. Considering that the tetramerization of protein building blocks occurs before the assembly, the tetramer containing mCherry will have less reactive sites and the incorporation will inevitably result in partial “quenching”. As this effect accumulates with increasing number of layers, the film growth will become slower at some point and the slope changes. Exposing the protein film to pulsed white LED light (30 klux) indeed led to stepwise release of mCherry-CarHC (Figure 4D). Nearly perfect synchronization between protein release and light illumination shows the swift photoresponsiveness of the LbL protein nanofilm (Figure 4E). The slight decrease in ΔF when light is off may arise from the readsorption of the cargos because of nonspecific hydrophobic interactions. The releasing effect upon pulsed light illumination or continuous illumination gives similar extent of release (Figure 4D). The linear correlation between the protein release and the duration of illumination points to the possibility of using this photoresponsive protein film for precise protein therapeutic delivery (Figure 4E). In addition, the total amounts of mCherry-CarHC proteins that are released from the protein nanofilms are related to the number of layers (Figure S8B). The thicker the film is, the more proteins that it releases, demonstrating its variable capacity. The release efficiency can be estimated based on the changes in frequency (see SI for details of calculation). It seems that for films of different number of layers, the release efficiency are all >90% (Table S1). The high release efficiency may be due to the fast transition kinetics of CarHC as well as the ultrathin nanolayer that presents little barrier for diffusion. In summary, we have successfully created a multistimuli responsive nanofilm on Au surface through the LbL assembly of engineered multidomain proteins comprising SpyTag/SpyCatcher pairs, thermally responsive ELP and photoresponsive CarHC. The protein tetramerization induced by AdoB12 in the dark turns out to be critical for the assembly. The resulting protein film is able to respond to multiple external stimuli such as temperature, divalent ions and light. It also enables controlled release of protein cargos in a light-dependent manner. The LbL assembly facilitated by protein oligomerization may represent a general strategy for creating responsive functional material surfaces.

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Wen-Bin Zhang: 0000-0002-8746-0792 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial supported from the National Natural Science Foundation of China to W.B.Z. (Grants 21474003 and 91427304) and from the Research Grants Council of Hong Kong SAR Government to F.S. (RGC-ECS Grant #26103915 and AoE/M-09/12).

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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/acsmacrolett.8b00147. Molecular cloning, protein expression and purification protocols, protein sequences, and other characterization of protein nanofilms (PDF).

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REFERENCES

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*Tel.: + 86 10 6276 6876. Fax: + 86 10 6275 1710. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Fei Sun: 0000-0002-3065-7471 517

DOI: 10.1021/acsmacrolett.8b00147 ACS Macro Lett. 2018, 7, 514−518

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DOI: 10.1021/acsmacrolett.8b00147 ACS Macro Lett. 2018, 7, 514−518