Expression and purification of TrCBM1
Escherichia coli BL21 (DE3) was used to express a histidine (His) tag–TrCBM1–green fluorescent protein (GFP) fusion protein. To obtain TrCBM1, the His tag and GFP regions were removed by adding enterokinase and thrombin, respectively. In our previous research, to analyze the molecular mass and conformation of 15N-labeled TrCBM1, we performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and 2D 1H–15N heteronuclear single-quantum correlation (HSQC) NMR [22]. In this work, SDS-PAGE and MALDI-TOF-MS were performed to characterize unlabeled TrCBM1. Pure TrCBM1 is a single protein with a molecular mass of 5195.8 Da. The MALDI-TOF-MS spectrum of TrCBM1 and a full-length SDS-PAGE gel are shown in Figure S1 of the Additional file.
Synthesis and characterization of the lignin models
Recently, we synthesized unlabeled β-O-4 lignin oligomer model compound 4 (Figure 2); its NMR signals were completely assigned to all carbon atoms and nonexchangeable protons by conducting 1D 1H NMR, 1D 13C NMR, 2D 1H–13C HSQC, 2D 1H–13C heteronuclear multiple bond correlation (HMBC), and 2D 1H–13C long-range heteronuclear single-quantum multiple bond correlation (LR-HSQMBC) experiments [25]. We used the protocol to synthesize three 13C-labeled β-O-4 lignin oligomer model compounds. The 13C labels were placed in the aromatic rings and β positions of compound 4(Arβ), α positions of compound 4(α), and methoxy groups of compound 4(m) using 13C-labeled vanillins and t-butyl-2-bromoacetate. The 13C-vanillins 1(Arβ), 1(α), and 1(m) were individually refluxed in acetone with unlabeled or 13C-labeled t-butyl-2-bromoacetate to afford 13C-t-butoxycarbonylmethyl vanillins 2(Arβ), 2(α), and 2(m), respectively. The 13C-t-butoxycarbonylmethyl vanillins were dissolved in anhydrous tetrahydrofuran (THF) and polymerized using lithium diisopropylamide (LDA). The ester groups in polymerized 13C-oligomers 3(Arβ), 3(α), and 3(m) were reduced using NaBH4 to obtain 13C-labeled β-O-4 lignin oligomer model compounds 4(Arβ), 4(α), and 4(m), respectively.
Figure 3 shows the 2D 1H–13C HSQC spectra of the 13C-labeled lignin models. Recently, we reported the HSQC spectra of the unlabeled lignin model [25]. A comparison of the HSQC spectra of the 13C-labeled and unlabeled lignin models indicated that the HSQC signal intensity was significantly enhanced by the incorporation of 13C, unequivocally demonstrating that 13C was incorporated into the designated positions in the labeled model. No HSQC signals attributable to the side products were observed in the NMR spectra, suggesting the fabrication of high-purity lignin models. The HSQC signals at δC/δH 85.9/4.52–4.64, 72.6/4.06–4.08, and 113.2–123.6/6.81–7.06 ppm in the HSQC spectra of lignin model 4(Arβ) with 13C labeling in the β positions and aromatic rings (Figure 3a) were assigned to the β positions, Terminal C1, and aromatic regions, respectively. The HSQC spectra of lignin model 4(α) (Figure 3b) contained three signals at δC/δH 74.7–75.0/4.92–4.98, 74.8–75.0/4.72–4.76, and 66.2/4.43–4.49 ppm, corresponding to the α positions in the A, B, and C rings, respectively. A single signal was observed at 58.1–58.3/3.57–3.85 ppm in the HSQC spectra of lignin model 4(m) with 13C-labeled methoxy groups (Figure 3c).
The long- and short-chain lignin models were separated via silica gel chromatography by sequential elution with (1) ethyl acetate, (2) ethyl acetate/methanol (5:1, v/v), and (3) ethyl acetate/methanol (2:1, v/v) for NMR and binding analyses. Size exclusion chromatography (SEC) revealed that the 13C-labeled lignin models exhibited narrow molecular weight distributions (Figure S2, Additional file). The degree of polymerization (DP) was calculated for each model compound based on its weight-average molecular weight (Mw). The DPs of the long- and short-chain lignin models ranged from 4.16 to 4.70 and from 2.64 to 3.12, respectively (Table 1). Although differences between the Mws of the long- and short-chain models were small, their NMR spectra (Figure 3d) and SEC profiles (Figure S2, Additional file) clearly differed. Therefore, the long- and short-chain lignin models could be used to evaluate the effects of Mw on molecular interactions with TrCBM1.
Table1 Molecular weight parameters of 13C-labeled and unlabeled β-O-4 lignin oligomer model compounds.
Lignin models
|
Chain length
|
Mn
|
Mw
|
Mw/Mn
|
DPa
|
Compound 4(Arβ)
(13C-labeled aromatic rings and β positions)
|
Long
|
843
|
923
|
1.095
|
4.70
|
Short
|
480
|
520
|
1.083
|
2.64
|
Compound 4(α)
(13C-labeled α positions)
|
Long
|
750
|
918
|
1.224
|
4.67
|
Short
|
531
|
614
|
1.156
|
3.12
|
Compound 4(m)
(13C-labeled methoxy groups)
|
Long
|
629
|
818
|
1.301
|
4.16
|
Short
|
482
|
587
|
1.218
|
2.98
|
Compound 4
(unlabeled)
|
Long
|
882
|
964
|
1.092
|
4.91
|
Short
|
552
|
560
|
1.014
|
2.85
|
a DP: Degree of polymerization calculated from the Mw and theoretical molecular mass of the lignin model.
Analysis of interactions between lignin models and TrCBM1
The interactions between the lignin models and TrCBM1 were evaluated via CSP analysis using 2D 1H–13C HSQC NMR. To observe the NMR signal perturbation with high clarity, the concentration of TrCBM1 should be several times higher than that of the lignin model. However, owing to experimental limitations for preparing a large amount of high-purity TrCBM1, we used the 13C-labeled lignin model to decrease the concentration of the additive. The lignin models are composed of two terminal units (A and C rings) and internal units (B rings), which are solely interlinked via the β-O-4 linkages with the erythro configuration. In the synthesized lignin model, only one alignment of the A and B rings was found, although the C ring exhibited two different alignments designated as C(I) and C(II) [25]. In 90% D2O, both configurations adopted the folded conformation; however, C(II) had a slightly more compact conformation than C(I) [25].
The 2D 1H–13C HSQC spectra of the lignin models (50 µM) in the presence and absence of TrCBM1 were superimposed (Figure 4). Several HSQC signals acquired in the presence of 200 µM TrCBM1 showed obvious perturbation, particularly those in the aromatic regions in the long-chain lignin spectra. Most of these signals exhibited larger perturbations when the concentration of TrCBM1 was increased to 350 µM. The observed perturbations in the aromatic ring are shown in a close-up view (Figure 4b), and these clear perturbations are good indicators of the interaction [26]. Conversely, the NMR signals obtained from the aliphatic regions and methoxy groups in the presence of TrCBM1 showed no significant perturbation (Figure 4c). These results clearly indicated that the aromatic regions in the long-chain lignin models were the primary sites for interactions with TrCBM1. However, no distinct perturbations were observed in the signals acquired from the methoxy groups or any of the aromatic and aliphatic regions when the short-chain lignin models were used for CSP analysis (Figures S3, Additional file). This indicated that the length of the lignin chains was an important factor in TrCBM1 binding.
The change in the chemical shift (Δδ) of each signal was calculated by subtracting the chemical shift value in the spectrum of the lignin model from that of the model recorded in the presence of TrCBM1. The 13C–13C coupling was not eliminated in the HSQC spectra. The CSP analysis was performed by tracing the center of the 13C–13C coupling signals with high accuracy because each signal was distinct in the HSQC spectra. The Δδ values obtained on the 1H and 13C axes are plotted in Figure 5. The Δδ values were large only for the signals obtained from the aromatic regions in the long-chain lignin models, whereas those were small for the signals acquired from the aliphatic regions and methoxy groups in both the long- and short-chain lignin models. The Δδvalues of the A 2, C 2(I), B 5, C 5(I), C 5(II), A 6, C 6(I), and C 6(II) signals obtained from the aromatic regions of the long-chain lignin models were all larger than 0.006 ppm on the 1H axis, whereas the Δδ values of the A 2 and C 5(I) signals on the 13C axis were greater than 0.05 ppm (Figure 5a). For the short-chain lignin models, only the Δδ values of the B 5 signals on the 1H axis exceeded 0.006 ppm (Figure 5b). Line broadening was observed in the B 5 signals of the long-chain lignin models in the presence of TrCBM1 (350 µM). The observed line broadening could be attributed to both the on and off rates of the complex formation and multiple binding states of lignin due to nonspecific binding [22].
Adsorption experiments with the lignin models
The unlabeled long- and short-chain lignin models of compound 4 were used to evaluate TrCBM1 binding affinity based on the Langmuir adsorption model. The Mws and molecular weight distributions of the unlabeled lignin models (Table 1) were nearly identical to those of the 13C-labeled lignin models (Figure S2, Additional file). The TrCBM1–His tag fusion protein was used instead of TrCBM1 without a His tag to conduct the adsorption experiments because the soluble lignin models could not be separated from the TrCBM1 protein via centrifugation. After incubating the sample solutions containing the lignin models and TrCBM1–His tag at 50°C for 1 h, cOmplete His-Tag Purification Resin (Roche, Basel, Switzerland) was added to the solutions. The unbound lignin models in the supernatant could then be separated from the bound lignin models, which were adsorbed on the precipitated TrCBM1–His tag that was bound to the His tag resin. By performing control experiments, we confirmed that all of the TrCBM1–His tag could adsorb onto the His tag resin. No undesired binding was observed between the His tag resin and lignin models (Figures S6 and S7, Additional file). To calculate the adsorption parameters summarized in Table 2, the concentrations of the unbound lignin models in the supernatants were determined. Although the long- and short-chain lignin models showed similar amount of the lignin model bound to His tag–TrCBM1 at saturation (Γmax), the Langmuir affinity constant (KL) of the long-chain lignin model was eight times higher than that of the short-chain lignin model. This result was consistent with that of the NMR interaction analysis. The percentages of the lignin models that bound to TrCBM1 were calculated using the KL values. In the CSP analysis, 84.6% and 91.4% of the long-chain lignin models were bound to TrCBM1 at TrCBM1 concentrations of 200 and 350 µM, respectively. At TrCBM1 concentrations of 200 and 350 µM, 33.0% and 46.6% of the short-chain lignin models, respectively, were bound to TrCBM1. The chain length was thus an essential factor in the binding between TrCBM1 and the lignin chains, which exclusively contained β-O-4 linkages. We also found that a DP of greater than 4 was required for significant adsorption.
Table 2 TrCBM1–His tag adsorption parameters of the unlabeled lignin models based on the Langmuir adsorption model.
Lignin model
|
Langmuir affinity constant, KL
(mL/mg)
|
Amount of adsorption at saturation, Γmax
(µg/mg)
|
Long chain
|
36.26
|
8.77
|
Short chain
|
4.47
|
9.15
|