Letter Cite This: ACS Macro Lett. 2018, 7, 1388−1393
pubs.acs.org/macroletters
Controlling SpyTag/SpyCatcher Reactivity via Redox-Gated Conformational Restriction Wen-Hao Wu,† Jingjing Wei,‡ and Wen-Bin Zhang*,† †
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 ‡ College of Chemical and Environmental Engineering, Anyang Institute of Technology, Anyang, Henan 455000, P. R. China
ACS Macro Lett. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 11/13/18. For personal use only.
S Supporting Information *
ABSTRACT: Herein, we report that the reactivity of genetically encoded SpyTag/SpyCatcher chemistry can be manipulated via redox-gated conformational restriction, which facilitates the preparation of all-protein-based hydrogel with latent reactive sites for subsequent covalent functionalization.
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conformation of SpyTag may offer a convenient way to manipulate its reactivity. In this letter, we report a SpyTag variant, SpyTagGB1, whose reactivity can be gated by redoxresponsive conformational restriction (Scheme 1), and show its utility in the making and sequential decorating of allprotein-based hydrogels (Scheme 2).
ontrolling reactivity is central in chemistry. For functional groups involved in most organic reactions, their reactivities are mostly defined by the corresponding chemical structures with explicit configuration and energy level of valence electrons, as well as the steric hindrance from neighboring groups. The recent emergence of genetically encoded “peptide−protein” chemistry embraces a new paradigm of encrypting chemical reactivity in protein sequences.1−4 The coding can be very different for reactive pairs developed from different ancestor domains, such as isopeptide-N/pilin-N, 5 SpyTag/SpyCatcher,6 SnoopTag/ SnoopCatcher,7 and SdyTag/SdyCatcher pairs.8 Even for the SpyTag/SpyCatcher reaction, the reactivity space can be dramatically expanded via mutation,9,10 deletion,11 circular permutation and insertion,12 or further splitting13 to impart mutually orthogonal reactivity, pH-responsive reactivity, cleavable feature, etc. Compared to conventional bioconjugation methods such as bioorthogonal click reactions,14 native chemical ligation,15 and various ligation techniques mediated by split intein,16 sortase A,17 or butelase 1,18 this class of “molecular superglue” techniques is unique in that they are fully genetically encodable, generate side-chain cross-links, and are usually highly efficient and specific in cellular environments. As a result, they have received extensive interest in various contexts, such as engineering synthetic vaccines,19 preparing all-protein-based hydrogels,20 controlling protein cellular locations,21 and designing protein backbone topology.22−24 Nonetheless, it remains a challenge to control its “spring-loaded” reactivity on demand, which could be achieved by gating with certain stimuli. Considering that the transfer of chemical information from sequence to reaction is warranted by protein folding, we hypothesized that controlling the © XXXX American Chemical Society
Scheme 1. Redox-Gated Conformational Restriction for Reactivity Control of the SpyTag/SpyCatcher Reactiona
a
The model of SpyTagGB1 was predicted by I-tasser25 based on the structure of GB1 (PDB: 5JXV), and the model of the SpyTag/ SpyCatcher complex was based on PDB: 4MLS.
Received: September 1, 2018 Accepted: November 7, 2018
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DOI: 10.1021/acsmacrolett.8b00668 ACS Macro Lett. 2018, 7, 1388−1393
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ACS Macro Letters
variant with SpyTag sequence embedded in the second loop region between the residues 45 and 46 and mutated residues 42 and 46 to cysteines for conformational lock by disulfide bonds. The amino acid sequence of this variant, SpyTagGB1 (or AGB1), in alignment to GB1 is shown in Figure S1. We envisioned that when expressed and oxidized, SpyTagGB1 has a conformation unfavorable for reaction with SpyCatcher due to confinement within the loop, and under reducing environments, the transiently unfolded species will be captured by SpyCatcher as driven by the formation of a stable covalent complex, giving selectivity and reactivity control. The gene of SpyTagGB1 was synthesized, amplified, and cloned into the pQE80L vector for expression in E. coli. Origami B (DE3) strain (see Figure S2 for all protein sequences). After affinity purification, it was characterized by SEC, SDS-PAGE, LC-MS, MALDI-TOF mass spectrometry, and CD spectrometry. As shown in Figure 1, SDS-PAGE and LC-MS spectrum confirm that the SpyTagGB1 has the expected molecular weight. Its CD spectrum also exhibits the characteristic peaks of α/β structure similar to that of GB1, indicating a folded structure. The decrease in the CD signal at ∼215 nm suggests that the insertion of SpyTag does disturb the β sheet region of GB1 to a certain extent, which is consistent with a previous report on similar cases.31 The SEC trace of the crude products of SpyTagGB1 exhibits shoulder peaks at low retention volumes (Figure S3), which are probably domain-swapped oligomers.32 The main peak corresponding to monomers was isolated for model reactions. For simplicity, SpyTag is denoted by “A”, and SpyCatcher is denoted by “B” in the following discussions. The redox-gated reactivity of SpyTagGB1 (AGB1) was examined by its reaction with B and B-GFP (Figure 1). The
Scheme 2. Sequential Site-Selective Functionalization of the Spy-Network Hydrogel
Protein folding governs the conformation and chemical environments essential for reactivity control. For SpyTag, conformational restriction can be easily achieved by confining it within the loop region of a folded protein. The B1 immunoglobulin G binding domain of streptococcal protein G (GB1) is a small artificial elastomeric protein with unique traits including fast folding kinetics, high folding fidelity, and robust folding.26−28 It has been shown that the second loop of its mutant GB1-L5 could tolerate the insertion of I27w34f protein to give a mutually exclusive protein GB1-L5/I27w34f.29 Aided by disulfide bond formation, the rapid folding of GB1-L5 kinetically traps the I27w34f protein in the unfolded state. Only under reducing environments, it gradually transits to the thermodynamically more stable folded I27w34f at the expense of GB1-L5 unfolding.30 Inspired by this, we designed a GB1
Figure 1. (a, b) SDS-PAGE analysis of SpyTagGB1 (AGB1) and its redox-gated reation with SpyCatcher (B) (a) and SpyCatcher-GFP (B-GFP) (b). (c) SEC overlay of AGB1 (black), B (red), and the reaction mixtures with (pink) or without 5 mM TCEP (blue). (d, e) LC-MS spectra of AGB1 (d) and the reaction mixtures of AGB1+B with 5 mM TCEP (e). (f) CD spectra of AGB1 (black), GB1 (red), and the ligated products of AGB1+B (blue) and A+B (pink). 1389
DOI: 10.1021/acsmacrolett.8b00668 ACS Macro Lett. 2018, 7, 1388−1393
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ACS Macro Letters two reactants were mixed in stoichiometric ratio (20 μM each) in PBS (pH = 7.4) at 37 °C for 1 h with 5 mM tris(2carboxyethyl)phosphine (TCEP). The control experiments were performed under identical conditions without TCEP. As shown in Figure 1a,b, the SDS-PAGE analysis clearly indicated that the ligated product only largely formed under reducing conditions with TCEP, and very little product was observed in the absence of TCEP especially for B-GFP owing to the not fully oxidized or misfolded AGB1. The results were corroborated by SEC (Figure 1c) and LC-MS characterizations (Figure 1d,e, Figures S4c, S5). The SEC trace of the reaction mixture without TCEP shows only one broad peak assigned as a simple mixture of both components, whereas the mixture with TCEP displays an apparent shift to the lower retention volume. LCMS further revealed a new molecular peak at 25419 Da, which matches well with the expected value of the ligated product (25414) (Figure 1e). Correspondingly, there is also a drastic change in the CD spectrum after reaction (Figure 1f). The typical CD signals of α/β structure of GB1 disappeared, and the pattern of the ligated product of AGB1 and B is similar to that of the CnaB233 and the SpyTag/SpyCatcher complex.12 It indicates that the reaction forms a well-folded SpyTag/ SpyCatcher complex, and the folded structure of GB1 is probably lost. Hence, AGB1 does show redox-gated reactivity to both SpyCatcher and the fusion protein, B-GFP. For a quantitative assessment on the reactivity under reducing environments, we further performed time course experiments at the same reaction condition but lower concentration (15 μM) of AGB1and B to determine the second-order rate constant. The yield reached ∼80% in one hour, and the second-order rate constant was measured to be (1.85 ± 0.28) × 102 M−1 s−1, which is about one-fifth that of the value for the wild-type SpyTag/SpyCatcher reaction (Figure S6). The next question is whether such redox-gated reactivity can be preserved upon fusion to other proteins. A multidomain protein, A-ELP-AGB1-ELP-A (AAGB1A), was designed where ELP stands for elastin-like polypeptide, a typical unstructured protein.34 The reaction between AAGB1A and SpyCatcher would afford triadduct (AAGB1A+3B) under reducing conditions upon nondiscriminative functionalization and yield siteselective diadduct (AAGB1A+2B) under nonreducing conditions which may be subjected to further functionalization to prepare the heterotriadduct (AAGB1A+2B+B-GFP) (Figure 2a). Indeed, the SDS-PAGE analysis clearly shows that the major product for the reaction in the presence of 5 mM TCEP has higher apparent molecular weight than that of the reaction in the absence of TCEP (Figure 2b). The SEC analysis is consistent with SDS-PAGE and shows that the product obtained without TCEP has an intermediate retention volume between that of AAGB1A and that of the product obtained with 5 mM TCEP (Figure S7). The MALDI-TOF mass spectrometry further confirms that the product under nonreducing conditions has the m/z of 56 861 corresponding to the expected value of AAGB1A+2B (56 872), and the product under reducing conditions has the m/z of 71 461, which is higher than the diadducts by exactly one SpyCatcher motif (Figure S8). The site-selective functionalization in fusion protein is thus successfully demonstrated. The addition of one more B-GFP was performed by reacting the diadduct and BGFP again under reducing and nonreducing conditions in PBS. It clearly shows that only in the presence of TCEP the heterotriadduct could be obtained as evidenced by the product band at much higher position in SDS-PAGE analysis (Figure
Figure 2. (a) Site-selective reaction between AAGB1A and B and the tandem sequential functionalization with B and B-GFP. (b, c) SDSPAGE analysis of (b) AAGB1A and B and their reactions with or without TCEP and (c) the products of AAGB1A and B and its further reaction with B-GFP with or without TCEP.
2c). Although selective reaction could also be achieved by using orthogonal SnoopTag/SnoopCatcher reaction7 or AY/ BVA mutants,9 the current approach is advantageous in its high reactivity and rapid reaction kinetics. Therefore, it is very promising for various biological applications where selectivity, efficiency, and responsiveness matter. In the following section, we will show its utility in the fabrication and functionalization of an all-protein-based hydrogel. Hydrogels are cross-linked networks of hydrophilic polymer chains holding a significant portion of water and have been widely used as an artificial extracellular matrix in tissue engineering owing to their similarity to biological tissues and high biocompatibility and readily tunable properties.35−37 To promote cell−matrix interactions, it is important to decorate the network with various biological cues, such as growth factors and hormones, in a spatiotemporally controlled fashion.38 Among numerous bioconjugation strategies developed to date, the SpyTag/SpyCatcher reaction is unique with regard to its unparalleled reactivity, high selectivity, and full compatibility with cellular environments.20,39−41 It has enabled the preparation of entirely protein-based hydrogels referred to as the Network of Spies.20,42 The macroscopic properties of this Network of Spies are fully genetically encodable, as manifested by the unique uranyl sequestration capability43 and light-responsive properties44 imparted by proteins. When other cross-linking methods are used for gelation, it facilitates the network functionalization under very mild conditions.45−47 We envisioned that the redox-gated SpyTagGB1 could allow one to “double dip” the superior SpyTag/SpyCatcher chemistry sequentially for both gelation and functionalization (Scheme 2). The hydrogel was conveniently prepared by mixing 15 wt % solution of AAGB1A and SpyCatcher-ELP-SpyCatcher-ELPSpyCatcher (or BBB) in a nominal 1:1 ratio between A and B.20 Considering that AGB1 cannot react with B in the absence of TCEP, it does not participate in network formation and serves as a latent functional group for subsequent functional1390
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after 36 h, and the latter exhibits a rapid and continuous release profile that seems to level off after 24 h. This is consistent with covalent immobilization of B-GFP in the former case. We envision that this method can be modularly applied to the functionalization with other proteins as well, providing a platform for tuning the biochemical and mechanical properties of the hydrogel. In summary, we have developed redox-gated SpyTagGB1/ SpyCatcher reaction by confining SpyTag within the second loop region of GB1 protein and locking the conformation by a disulfide bond. The SpyTagGB1 is nonreactive under nonreducing conditions but exhibits spring-loaded reactivity under reducing conditions. It has enabled site-selective functionalization of a multidomain protein and tandem sequential functionalization of all-protein-based hydrogels. It expands the toolbox of genetically encoded peptide−protein chemistry and opens up a new approach to reactivity control, which shall be useful for a broad range of applications in biomaterials, chemical biology, and synthetic biology.
ization under reducing conditions. Gelation occurs within several minutes and was left to cure overnight. The dynamic shear rheology experiments were performed to characterize the mechanical properties. The strain-sweep profile shows a linear viscoelastic region up to 80% strain, indicating that the gel was robust even under large deformation (Figure 3a). Then, a
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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.8b00668.
Figure 3. (a) Strain-sweep profile and (b) frequency-sweep profile of the Spy-Network hydrogel (15 wt %). (c) Images of the protein release at different times from the hydrogels functionalized with BGFP in the presence (bottom) or absence (top) of TCEP. (d) Release profile shown by the plot of the fluorescence intensity of the supernatant versus time. The error bars are the standard deviation of the data (n = 3).
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Molecular cloning, protein expression and purification protocols, protein sequences, and other characterizations of protein hydrogels (PDF)
AUTHOR INFORMATION
Corresponding Author
frequency-sweep experiment was performed at 1% strain within the linear viscoelastic range, which shows slight decrease in storage modulus (G′) from 2 to 0.8 kPa at lower frequency (Figure 3b). We speculated that the decrease might be caused by the shear-induced unfolding of GB1 motifs that did not form disulfide bonds. A similar phenomenon has been observed in the gels cross-linked by the reconstituted GB1 fragments.32 Nevertheless, the storage modulus is much larger than the loss modulus over the entire frequency range, which is consistent with the formation of a covalently cross-linked hydrogel. The B-GFP was used as a model protein for hydrogel decoration. Prior to functionalization, the hydrogels were incubated with B in PBS to quench any residual SpyTag to exclude its contribution. After rinsing with PBS to remove B, functionalization was performed by soaking the hydrogel in a solution of B-GFP in PBS with 5 mM TCEP. The control experiments were run without TCEP. Despite its relatively large size, B-GFP has no problem diffusing into the hydrogel. Both physical adsorption and chemical attachment may occur, which makes the gel green fluorescent. We anticipated that physically absorbed B-GFP would slowly diffuse out of the gel upon dialysis, whereas the chemically attached ones could not. To distinguish them, protein release assays were performed by immersing the functionalized hydrogels in 0.5 mL of PBS (pH = 7.4) at 37 °C and monitoring the fluorescence of the supernatant at specific time intervals. The fluorescence intensity could reflect the released amounts of B-GFP in supernatant (Figure 3c). As shown in Figure 3d, the release profiles were very different for the hydrogels functionalized with or without TCEP. The former shows little release even
*Tel.: +86 10 6276 6876. Fax: +86 10 6275 1710. E-mail:
[email protected]. ORCID
Wen-Bin Zhang: 0000-0002-8746-0792 Author Contributions
The manuscript was written through contributions of all authors. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation of China (Grants Nos. 21474003, 91427304) and “1000 Plan (Youth)”. The work is also sponsored by the Interdisciplinary Medicine Seed Fund of Peking University (Grant No. BMU2018MC001).
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