Combination of Click Chemistry and Enzymatic Ligation for Stable and Efficient Protein Immobilization for Single-Molecule Force Spectroscopy

• DOI: 10.31635/ccschem.021.202100779
• Publication Date: 2021-02-17T15:24:17Z
• Authors: Shengchao Shi; Ziyi Wang; Yibing Deng; Fang Tian; Qingsong Wu; Peng Zheng
• Keywords: Force Spectroscopy

Open AccessCCS ChemistryRESEARCH ARTICLE1 Feb 2022Combination of Click Chemistry and Enzymatic Ligation for Stable Efficient Protein Immobilization Single-Molecule Force Spectroscopy Shengchao Shi, Ziyi Wang, Yibing Deng, Fang Tian, Qingsong Wu Peng Zheng Shi State Key Laboratory Coordination Chemistry, Biomedicine Innovation Center (ChemBIC), School Chemical Engineering, Nanjing University, 210023 , Wang Deng Tian *Corresponding author: E-mail Address: [email protected] https://doi.org/10.31635/ccschem.021.202100779 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail immobilization is an essential method both basic applied research protein, covalent, site-specific attachment the most desirable strategy. Classic methods typically rely on a heterobifunctional cross-linker, such as N-hydroxysuccinimide (NHS)-linker-maleimide, or similar two-step process. It utilizes amino-reactive NHS thiol-reactive maleimide conjugate protein solid support. However, chemical susceptible hydrolysis during storage handling, reacts nonspecifically with all cysteines available in leading inconsistent result. To solve these problems, we have developed by combining strain-promoted azide–alkyne cycloaddition (SPAAC) click reaction OaAEP1(C247A)-based enzymatic ligation. The was demonstrated successful enhanced green fluorescent (eGFP), which visualized imaging. Moreover, correct folding stability immobilized were verified atomic force microscopy-based single-molecule spectroscopy (AFM-SMFS) measurement high success rate (89%). Finally, strength 1,2,3-triazole linkage from azide-dibenzocyclooctyne (DBCO)-based SPAAC quantified ultrahigh rupture <1.7 nN. Thus, this stable, efficient, can be used many challenging systems, especially SMFS studies. Download figure PowerPoint Introduction crucial characterization application proteins,1,2 study mechanism, detection, study.3–7 Among different methodologies, covalent bonding desirable, it offers reliable robust attachment. This strategy relies cross-linking reagent that connect support through amino carboxyl functional group surface lysines.1,8 Perhaps commonly glass (NHS) ester. notorious unintended hydrolysis. extra care must paid its repeated usages keep effective, result will obtained if handled inappropriately. Also, (Mal) cysteines/lysines interest (POI) uncontrolled way. overcome issues, combine OaAEP1-based ligation immobilization. chemistry attracted increasing attention biology, chemistry, engineering. water yield, has been labeling immobilization.9–11 direct requires modification incorporation nonnatural acids target limits yield application.12,13 ligase also purposes, sortase A butelase 1.14–16 bottlenecks are low efficiency poor accessibility enzymes.17 Recently, transpeptidase OaAEP1(C247A)-mediated polyprotein construction immobilization.18 Here, combined N3-dibenzocyclooctyne immobilize imaging unfolding experiments.18–20 chemicals easier handling results. quantified, showing above 1700 pN, provides general measuring stable proteins bonds chemistry. Experimental Methods All reagents purchased commercial suppliers accordingly. Imidazole-1-sulfonyl azide hydrochloride (ImSO2N3·HCl; Abydos Scientific, Nanjing, Jiangsu, China), DBCO-(polyethylene glycol)4-Mal (DBCO-PEG4-Mal; Biocone, Chengdu, Sichuan, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester, sodium salt (sulfo-SMCC; Thermo Fisher, Rockford, IL) stored dark. noted ImSO2N3·HCl hygroscopic slowly produce hydrazoic acid, sensitive explosive. Care taken prevent occurring. Microporous membrane filters (0.22 0.45 μm) further purification (Jet Biofil, Guangzhou, Guangdong, China). genes eGFP, Coh, XDoc, GB1, I27, SdrG, Fgβ Genscript (Nanjing, OaAEP1(C247A) cysteine 247 alanine mutant asparaginyl endoproteases 1 Oldenlandia affinis, abbreviated OaAEP1. I27 27th immunoglobulin domain human cardiac titin. GB1 B1 Streptococcal G.21 [Coh-XDoc] type III cohesion dockerin-X module complex Ruminococcus flavefaciens.22 SdrG serine-aspartic (SD)-repeat G, cell adhesin Staphylococcus epidermidis, N terminus β chain fibrinogen.23 overexpressed Escherichia coli BL21(DE3) cells. details found Supplementary Information. For AFM-SMFS experiment, Nanowizard4 AFM used. cantilevers (MLCT-Bio-DC; Bruker, Camarillo, CA) calibrated buffer thermal noise measure spring constant. experiments performed under constant pulling velocity 1000 nm/s. data first filtered using processing software (JPK, Berlin, Germany). More regarding selection analysis Supporting Results Discussion principle our shown Figure 1. First, replaces (Figures 1a 1b). Starting amino-silanized coverslip, N3 functionalized between –NH2 (step 1).24 Then, DBCO-PEGn-Mal reacted adds Mal 2). instead randomly reacting POI, peptide including elastin-like polypeptide (ELP)n spacer, glycine-leucine-ELP-cysteine (GL-ELP-C) 3).25 GL tag, recognized several transpeptidases, OaAEP1 Sortase A. These endopeptidases function ligases corresponding tag [asparagine-glycine-leucine (NGL) OaAEP1, leucine-proline-glutamic acid-threonine-glycine (LPETG) A] 4).18,26 | (a) classic uses sulfo-NHS-linker-Mal (SMCC) surface. (b) Our method: shelf-stable ImSO2N3 group. –N3, followed addition GL-ELP-C. POI NGL functionalized-GL catalyzed allow easy confirmation, eGFP model clear pattern slides 2a 2b Information Figures S1–S4). preparation protocol provided solution added GL-functionalized slide incubated hand pipetting. washed high-salt deionized remove nonspecific bound dried. air, showed edge formed covalently attached (Figure 2a). In addition, automatic microarrays machine, 3 × array air 2b). fluorescence image no difference dry sample ( S4a). Under same conditions, droplet without much weaker signal S4b). transformation amines analyzed azide-functionalized surfaces SMCC react unreacted amine last step control experiment. And subsequent steps method, only After quantification ImageJ (National Institutes Health, Bethesda, MD) normalization new 5% intensity per area indicating ∼95% (see 2c, Information, Table S1). 2 Verification measurement. Immobilized pipetting shows signal, whose boundary indicated dashed line bright-field S3). microarray, expected fluorescence. (c) three methods, SMCC, determining azide, normalized. histograms show (green) (black) (d) schematic how measures eGFP. (e) Force-extension curves signals (curves 2, green), while observed outside 4). (f) Diagram relationship F ΔLc (one-step green, n = 238; purple, 30) (in black). confirm manipulates single molecule27–31 complexes mechanically,32–34 (un)fold protein,35–38 break molecular interactions,39–41 chemically bond.42–48 previous design, cohesin (Coh) fused binds Xmod-dockerin (XDoc) site specifically reversibly ∼600 pN.2,49 tip GB1-XDoc marker contour length increment (ΔLc) 18 nm.21 By pressing glass, captured interaction. moving up vertically at (1000 nm/s), stretched unfolded 2d). ruptured, moved another place repeat probing droplet-covered AFM-SMFS, force-extension peaks observed. Fitting curve worm-like model, besides peak nm, additional ∼80 nm 2e, 1, green). agrees theoretical results full extension (227 acids). 96 ± 13 pN (average standard deviation, 238) 84 2f).50 scenario observed, findings GFP (curve We probed control. As expected, containing detected prove well-folded proper demonstrate ability large proteins, domain, I27. characterized ∼220 ∼28 (pulling speed: nm/s).35,51 built Coh-(GB1)2-(I27)8-NGL polyprotein, more than 10 domains. then experiment GB1-XDoc-functionalized 3a). Its sawtooth-like 11 peaks, 8 red) blue), respectively 3b). 228 32 (n 524) 212 37 217) 3c 3d). values comparable NHS(SMCC)-based S5). Statistically, 48 out 54 (89%) succeeded pick-up ratio (<1%). Coh-(GB1)2-(I27)8 method. unfolded. designed. colored red marked star, blue star. (c), (d), respectively. mechanical system, triazole N3-DBCO reaction, quantified. test high-force limit, protein–protein interaction pair [SdrG-Fgβ] over 2000 S6),23 pair. Fgβ-(GB1)2-NGL SdrG-NGL them system 4a). two (213 34 318, 4b 4c), detachment average 1717 185 313) 4d, star), could withstand forces ∼1700 pN. statistical value one strongest “click bond” measured so far.52–54 4 Schematic 1,2,3 complex. (blue star) (rupture) (red star). 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