Lignin Cross-Links with Cysteine- and Tyrosine-Containing Peptides

Article pubs.acs.org/JAFC

Lignin Cross-Links with Cysteine- and Tyrosine-Containing Peptides under Biomimetic Conditions Brett G. Diehl*,† and Nicole R. Brown‡ †

Department of Agricultural and Biological Engineering, 226 Forest Resources Building, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ‡ Department of Agricultural and Biological Engineering, 209 Agricultural Engineering Building, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *

ABSTRACT: The work presented here investigates the cross-linking of various nucleophilic amino acids with lignin under aqueous conditions, thus providing insight as to which amino acids might cross-link with lignin in planta. Lignin dehydrogenation polymer (DHP) was prepared in aqueous solutions that contained tripeptides with the general structure XGG, where X represents an amino acid with a nucleophilic side chain. Fourier-transform infrared spectroscopy and energy dispersive X-ray spectroscopy showed that peptides containing cysteine and tyrosine were incorporated into the DHP to form DHP−CGG and DHP−YGG adducts, whereas peptides containing other nucleophilic amino acids were not incorporated. Scanning electron microscopy showed that the physical morphology of DHP was altered by the presence of peptides in the aqueous solution, regardless of peptide incorporation into the DHP. Nuclear magnetic resonance (NMR) spectroscopy showed that cysteinecontaining peptide cross-linked with lignin at the lignin α-position, whereas in the case of the lignin−tyrosine adduct the exact cross-linking pathway could not be determined. This is the first study to use NMR to confirm cross-linking between lignin and peptides under biomimetic conditions. The results of this study may indicate the potential for lignin−protein linkage formation in planta, particularly between lignin and cysteine- and/or tyrosine-rich proteins. KEYWORDS: nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectroscopy, energy dispersive X-ray spectroscopy, scanning electron microscopy, lignin, peptide, cross-linking

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INTRODUCTION Lignin is an abundant, aromatic biopolymer that forms in the lignocellulosic matrices of plant cell walls. Its free radical polymerization mechanism and heterogeneous nature make it unique within the plant kingdom. Lignin is economically important to the pulp and paper industries, the agricultural industries, and the biofuels and biorenewables industries, all of which are hampered by its recalcitrance against extraction and/ or degradation.1−7 Many aspects of lignification are still poorly understood, despite its abundance and economic relevance. For example, the extent to which lignin interacts with surrounding cell wall polymers, particularly proteins, is largely unknown. It has been suggested that lignin forms covalent cross-links with plant cell wall components, particularly hemicelluloses.8−11 One prevalent pathway for lignin−carbohydrate linkage formation is thought to be through the reaction of a nucleophilic moiety (e.g., a hydroxyl or carboxylic acid group) with the electrophilic α-carbon of the lignin quinone methide (QM) intermediate.12,13 The cross-linking of lignin with other cell wall components, such as proteins, has not been well investigated, despite the fact that lignin−protein linkages may play important roles in wild type and transgenic plant lines. In most wild type plant lines the pattern of lignin deposition indicates the presence of so-called nucleation sites within specific regions of the plant cell wall (e.g., the cell corners), but the nature of these nucleation sites remains unknown.1 Nucleation sites may be rich in structural proteins, perhaps leading to lignin−protein cross-linking, but this hypothesis has © 2014 American Chemical Society

not been adequately tested. A recently engineered line of Populus secretes a tyrosine-rich peptide into the cell wall. Increased sugar extractability was observed in these Populus lines upon protease digestion of the walls, and it was hypothesized that this was due to lignin−protein linkage formation. However, the putative lignin−protein linkages have yet to be identified.14,15 Recently, Cong et al. used radioactive labeling and other techniques to show that proteins associate with lignin in vitro,16 and Diehl et al. showed that amino acids bearing nucleophilic side chains, namely, Cys, Lys, His, Asp, Glu, Ser, and Tyr, all react with a lignin model QM in dichloromethane.17 Diagnostic NMR shifts of lignin−peptide compounds were described, but the propensity for such linkages to form under biomimetic conditions (i.e., as lignin polymerizes with peptides in aqueous media) was not explored. To expand upon these results, we investigated the propensities for various tripeptides to cross-link with lignin as it was polymerized synthetically to afford lignin dehydrogenation polymer (DHP), which is a biomimetic lignin model compound.18 It is anticipated that this will assist in future studies to help elucidate the interactions between lignin and proteins in planta. Received: Revised: Accepted: Published: 10312

August 12, October 2, October 2, October 2,

2014 2014 2014 2014

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Figure 1. Lignin−peptide cross-linking pathway. Lignin−peptide cross-links form when nucleophilic side chains of amino acids react with quinone methides formed during lignin β-ether coupling. R = H or OMe, L = lignin.

useful toward the continued study of lignin formation in both native and mutant plant lines.

To investigate the propensity for lignin−peptide crosslinking under biomimetic conditions, lignin dehydrogenation polymer (DHP) was prepared in aqueous solutions containing tripeptides. Each tripeptide had the general structure X− glycine−glycine (XGG), with X being cysteine (C), lysine (K), histidine (H), aspartic acid (D), glutamic acid (E), serine (S), tyrosine (Y), threonine (T), or hydroxyproline (Hyp). These amino acids were previously identified as being reactive (or potentially reactive in the case of T and Hyp) toward lignin QMs.17 The general peptide structure and predicted mode of lignin−peptide cross-linking are shown in Figure 1. The Ctermini and N-termini of the peptides were blocked via amidation and acetylation, respectively, to ensure that the amino acid of interest (i.e., residue X) contained the only nucleophilic moiety. Glycine was chosen as the “place holder” residue due to its expected lack of reactivity toward lignin. The lengths of the peptides were limited to three residues because in vitro reactions of large peptides with DHPs result in the formation of lignin−peptide complexes that are insoluble and thus difficult to characterize (e.g., liquid state NMR becomes impractical) (results not shown). Tripeptides were added in 25% mol/mol ratio to the lignin monomer (coniferyl alcohol) because it was previously reported that lignin DHPs contain between 20 and 30% β-ether linkages.19 Thus, the ratio of nucleophilic amino acids to lignin β-ether QMs was expected to be approximately 1:1 over the course of the polymerization reaction. Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and nuclear magnetic resonance spectroscopy (NMR) were used to characterize the lignin−peptide adducts. FT-IR and, more recently, NMR, have become staples of lignin characterization.20−22 Multidimensional NMR techniques (e.g., heteronuclear single quantum coherence (HSQC)) are particularly useful because the shift degeneracies observed in 1D spectra are largely eliminated. Furthermore, diagnostic NMR shifts of lignin−peptide model compounds have previously been assigned.17 SEM imaging of synthetic and native lignins has not garnered much research attention,23,24 but the technique was employed here to visualize morphological differences between neat DHP and the lignin−peptide adducts. It was useful to also collect EDS elemental analysis data while the lignin−peptide samples were in the SEM instrument, with the presence of nitrogen suggesting peptide incorporation. Through the use of these techniques this study provides new insights into the propensities and mechanisms of lignin−peptide linkage formation. It is expected that this will be

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MATERIALS AND METHODS

Materials. All chemicals necessary for DHP preparation were purchased from Sigma with the exception of the peptides (>95% purity), which were purchased from Peptide 2.0 (www.peptide2.com). The peptides acquired were of the sequence XGG, where X represents one of the following: cysteine (C), lysine (K), histidine (H), aspartic acid (D), glutamic acid (E), serine (S), tyrosine (Y), threonine (T), or hydroxyproline (Hyp). Synthesis of Lignin DHP and Lignin−Peptide Adducts. Guaiacyl-based DHP was synthesized in sodium phosphate buffer (pH 6.5) using coniferyl alcohol as the sole lignin monomer.18 The DHP crude product was centrifuged (10000g, 20 min, 4 °C) and the pellet washed four times with distilled water. The DHP product was then lyophilized to yield dry DHP (typical yields 60−70%), which was characterized via NMR as described below and was found to contain shifts typical of G−DHP.20,22 Lignin−peptide adducts were prepared as above, with the exception that 25% peptide to coniferyl alcohol (mol/mol basis, all tripeptides were freely water-soluble at the concentrations employed) was added to the flask containing coniferyl alcohol prior to the start of the reaction. The crude reaction products were centrifuged, washed four times, and lyophilized as described above to yield tan powders. Products were stored at −40 °C and characterized using FT-IR, SEM, EDS, and NMR. Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy. SEM images were collected on a field emission SEM (FESEM-FEI NanoSEM 630) at 2 or 3 kV under high vacuum (1.7 × 10−6 Torr). Samples were not sputter-coated prior to imaging. Characteristic X-rays were collected with an X-Max silicon drift detector (Oxford Instruments) at 10 kV under low-vacuum conditions (0.6 Torr) to prevent sample charging. Elements were selected and quantified using Aztec Energy Analyzer Software (Oxford Instruments). Three locations were analyzed per adduct, and average atomic percentages and standard deviations are reported under Results and Discussion. Nuclear Magnetic Resonance Spectroscopy. The neat peptides (25 mg) were dissolved in DMSO-d6/pyridine-d5 (4:1 v/v, 500 μL), and proton (16 scans), carbon (4000 scans), HMQC (64 scans), and HMBC (32 scans) spectra were collected using standard Bruker pulse programs on a Bruker DRX-400 (400 MHz 1H resonance frequency) using the central solvent peak [δH/δC, dimethyl sulfoxide (DMSO), 2.50/39.50 ppm] as internal standard. In the case of DHP and the lignin−peptide adducts, NMR spectra were acquired on a Bruker Biospin (Billerica, MA, USA) AVANCE 500 (500 MHz 1H resonance frequency) spectrometer fitted with a cryogenically cooled gradient probe having inverse geometry, that is, with the proton coils closest to the sample. Spectra were processed with Bruker’s Topspin 3.1 software, using the central solvent peak as internal reference [δH/ δC, dimethyl sulfoxide (DMSO), 2.50/39.5 ppm]. The DHP or lignin−peptide adducts (∼45 mg) were placed in an NMR tube (i.d. = 10313

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4.1 mm), dissolved in DMSO-d6/pyridine-d5 (4:1 v/v, 500 μL), and subjected to adiabatic HSQC (‘hsqcetgpsisp2.2’) experiment. In the case of DHP−YGG, HMBC (“hmbcgpndqf”), COSY (“cosygpqf”), and NOESY (“noesyesgpph”) experiments were also conducted to attempt to determine the lignin−tyrosine cross-linking mechanism. Processing used typical matched Gaussian apodization in F2 (LB = −0.3, GB = 0.001), squared cosine-bell, and one level of linear prediction (32 coefficients) in F1.25 For an estimation of the various interunit linkage types in DHP and lignin−peptide adducts, the wellresolved Cα−Hα contours were integrated; no correction factors were used. Fourier-Transform Infrared Spectroscopy. Lignin DHP, neat peptides, and lignin−peptide adducts were analyzed using a Bruker Vertex V70 spectrometer (Bruker Optics) equipped with an MVP-Pro diamond single reflection ATR accessory (Harrick Scientific), and 100 scans at 6 cm−1 resolution were averaged for each sample using a DTGS detector and a scan frequency of 5 kHz. In all cases, the spectrum of the clean diamond crystal was used as the reference spectrum. All spectral manipulations were performed using OPUS 6.0 (Bruker Optics).

Lignin−Peptide Morphology. SEM was used to compare the morphologies of neat DHP and the lignin−peptide coproducts (Figure 2). The neat DHP particles clumped together to form nearly perfect spheres, as reported previously.23,26 Comparatively, spheres of lignin−peptide adducts tended to form large, aggregate domains. This alteration of morphology was observed regardless of whether the peptide in question was covalently incorporated into the lignin (e.g., see DHP−DGG in Figure 2 and in Figure S2 in the Supporting Information). This change in morphology was most likely the result of favorable interactions with water; that is, the more hydrophilic tripeptides surrounded the more hydrophobic lignin particles, as would be expected in a heterogeneous polymerization. Further research is necessary to determine the influence of noncovalent interpolymer interactions during lignin polymerization. Lignin−Peptide Linkage Identification. Figure 3 shows the heteronuclear single-quantum coherence (HSQC) spectrum of DHP−CGG. This 2D NMR technique is particularly useful for lignin analysis because the shift degeneracy observed in 1D NMR spectra is largely avoided. Novel shifts are shown in green, red, and blue, whereas standard lignin shifts are shown in black.20,21 Reference shifts of neat CGG (purple) were added to the spectrum during processing; these shifts were not observed in the DHP−CGG spectrum. Some peptide shifts migrated as a result of DHP−CGG cross-linking. For example, the cys-13C/1Hα shift (originally at 4.5/56.0 ppm in neat CGG) migrated to 4.6/52.8 ppm and the cys-13C/1Hβ shift (originally at 2.8/26.6 ppm in neat CGG) migrated to 2.8/32.9 ppm in the DHP−CGG adduct. These shifts migrated upon lignin− peptide cross-linking due to their proximity to the thiol group, which is the reactive center of the CGG peptide. Shifts of proton and carbon atoms located far from the reactive thiol were largely unaffected by cross-linking (e.g., shifts at 3.9/42.8 and 3.8/42.5 ppm). Two novel lignin shifts, found at 4.4/50.1 ppm (Figure 4, red peak) and 4.8/81.3 ppm (Figure 4, blue peak), confirmed covalent cross-linking of DHP with CGG. Similar lignin α-shifts (4.3/50.4 ppm) and β-shifts (4.7/81.7 ppm) were previously reported in a model compound study wherein a single cysteine residue was reacted with a lignin model quinone methide.17 The minute differences in shift locations can be attributed to changes in chemical environment between a small lignin model compound and a high molecular weight lignin. Volume integration of the HSQC contours showed that approximately 33% of the β-ether linkages in DHP−CGG exhibited cysteine functionality at the α-carbon, whereas the remaining β-ether linkages exhibited typical α-hydroxyl functionality and a minor fraction of α-aryl ether (α-O-aryl) moieties. This indicated that cysteine was an efficient trapper of lignin QMs under biomimetic conditions. Figure 4 shows the HSQC spectrum of DHP−YGG. As with the DHP−CGG adduct, incorporation of YGG peptide into lignin was evidenced by the appearance of diagnostic chemical shifts (Figure 4, green and orange contours). Reference shifts of neat YGG (purple, solid yellow, and solid orange contours) were added during processing. Several factors confound evidence for cross-linking between tyrosine-based peptide (YGG) with lignin. The structural similarity of tyrosine and the lignin monomer, coniferyl alcohol, led to substantial degeneracy in the NMR spectra, as discussed in greater detail below. In addition, the cross-linking of YGG with DHP may have occurred via two mechanisms. The first

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RESULTS AND DISCUSSION Preparation and Yields of the Lignin−Peptide Adducts. Lignin DHP was prepared in aqueous solutions containing water-soluble tripeptides (25% peptide/coniferyl alcohol mol/mol basis). Each tripeptide contained one nucleophilic amino acid and blocked N- and C-termini to mimic inclusion within a larger protein and to prohibit potential side reactions. The lignin−peptide adducts were collected via centrifugation, washed, and characterized via FTIR, SEM, EDS, and NMR. Water-soluble residues that remained in the supernatants following centrifugation and adduct collection were not analyzed. The results, detailed below, indicate covalent incorporation of CGG and YGG peptides into the lignin polymer, whereas other peptides did not show significant reactivity. Yields for the DHP and lignin−peptide adducts are shown in Table 1. Yields were determined by dividing the mass of Table 1. Yield Data for the DHP and Lignin−Peptide Adducts CAa (mg)

peptide (mg)

yield (mg)

yield (%)

200.0 200.0 200.0 200.0 200.0 200.0 200.0 200.0 200.0 200.0

0.0 88.4 83.6 86.1 80.0 83.9 72.2 93.3 76.1 79.4

130.0 100.0 174.5 176.0 180.0 197.9 172.8 114.2 179.1 177.4

65.0 34.7 61.5 61.5 64.3 69.7 63.5 38.9 64.9 63.5

DHP DHP−CGG DHP−KGG DHP−HGG DHP−DGG DHP−EGG DHP−SGG DHP−YGG DHP−TGG DHP−HypGG a

CA, coniferyl alcohol

recovered solids by the total starting mass (i.e., combined mass of lignin monomer and peptide). The yield of the DHP control was typical.18 In the cases of CGG and YGG (which we show below to be considerably reactive toward lignin), the yields of recoverable DHP were depressed. In these cases, the authors perceived that a portion of the lignin−peptide adducts may have been aqueous soluble and thus held in solution during the centrifugation process. Characterizations of these aqueoussoluble fractions was not carried out. 10314

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Figure 2. SEM images of DHP (top left), DHP−CGG (top right), DHP−YGG (bottom left), and DHP−DGG (bottom right). Scale bar = 2 μm. Refer to the Supporting Information for SEM images of other lignin−peptide adducts, which displayed morphologies similar to those of the DHP− DGG adduct.

Figure 3. Side-chain and aromatic regions (inset) of the HSQC NMR spectrum of DHP−CGG. Black shifts are typical of G−DHPs, green shifts correspond to peptide α- and β-signals, and red and blue shifts correspond to lignin α- and β-signals in β-ether/α-cysteine structures (top left). Purple shifts were added during processing to indicate shifts of neat CGG peptide.

potential mechanism involves oxidation of the phenolic hydroxyl of tyrosine by horseradish peroxidase, followed by recombination of the tyrosine radical with a radical on the lignin polymer. Previous work relied upon stop-flow kinetics to show that this mechanism is probably unfavorable.16 Still, we investigated the possibility of this compound forming by conducting heteronuclear multiple bond correlation (HMBC), correlation spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY) (data not shown). However, we

were unable to conclusively assign NMR shifts of lignin− tyrosine linkages formed in this manner. Either the mechanism is not valid under our experimental conditions and/or the shift degeneracy between lignin−tyrosine linkages and typical lignin shifts is too great to allow necessary spectral resolution. A second cross-linking mechanism between lignin and tyrosine is also possible if the phenolic hydroxyl of tyrosine quenches the lignin quinone methide to form the α-aryl ether structure shown in Figure 4. Importantly, phenolic hydroxyls of 10315

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Figure 4. Side-chain and aromatic regions (inset) of the HSQC NMR spectrum of DHP−YGG. Black shifts are typical of G−DHPs, green shifts correspond to peptide α- and β-signals, and red and blue shifts correspond to lignin α- and β-signals in β-ether/α-tyrosine structures (top left) and/ or lignin−lignin α-O-aryl structures. Purple shifts were added during processing to indicate shifts of neat YGG peptide. Within the aromatic region, solid yellow and orange shifts (added during processing) were assigned to the aromatic ring of tyrosine in neat YGG.

Figure 5. Side-chain region of the HSQC NMR spectrum of DHP−HGG. Black shifts are typical of G−DHPs, green shifts correspond to peptide αand β-signals, and red and blue shifts correspond to lignin α- and β-signals in β-ether/α-histidine structures (top left). Purple shifts were added during processing to indicate shifts of neat HGG peptide.

work,16 as well as the NMR, IR, and EDS data (shown below), strongly suggests that the YGG peptide cross-linked with lignin DHP via quenching the quinone methide; however, diagnostic evidence supporting this remains elusive. Peptide peaks could always be observed when the HSQC contours were viewed quite low (i.e., near the signal-to-noise limit), even for lignin−peptide adducts other than DHP−CGG and DHP−YGG. Figure 5 shows the HSQC spectrum of DHP−HGG. This sample showed the highest concentration of peptide after DHP−CGG and DHP−YGG. A putative lignin−α-histidine cross-link was observed at 5.7/60.4 ppm, in good agreement with the α-shift of a lignin−histidine model compound (5.7/60.2 ppm).17 Volume integration showed that the lignin−α-histidine shift accounted for only ∼0.1% of the total lignin α-signal. It is noteworthy that this low abundance of peptide was detected by HSQC NMR but not readily detected by IR or EDS, thus illustrating the sensitivity of multidimensional NMR toward investigating lignin−protein linkages.

the monolignols could participate in the same reaction. Given the similarity of these structures, shift degeneracy again complicates the investigation of the mechanism, as a lignin− tyrosine model compound exhibited similar NMR shifts (α-1H/13C, 5.5/78.3 ppm in DMSO/pyridine) to α-aryl ether linkages known to occur in neat coniferyl alcohol-based DHPs (α-13C, 79.01 ppm in DMSO).17,27 In an attempt to overcome this issue, the well-resolved HSQC α-signals of neat DHP and DHP−YGG adduct were integrated. It was observed that α-aryl ether shifts comprised approximately 4.2% of the total α-signal in DHP−YGG but only 1.9% in neat DHP synthesized under similar conditions. This increase could be due to cross-linking, but other factors exist as well, such as imprecision in the HSQC volume integration or random variation among DHP syntheses (other lignin−peptide adducts displayed similarly high α-aryl ether signals), making it unclear if the structure shown in Figure 4 formed in the DHP−YGG adduct. In summary, previous 10316

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Table 2. Interunit Linkage Ratios of the DHP and Lignin−Peptide Adductsa HSQC signal ratios DHP DHP−CGG DHP−DGG DHP−EGG DHP−KGG DHP−HGG DHP−SGG DHP−YGG DHP−TGG DHP−HypGG

β-ether/α-OH

β-ether/α-O-aryl

β-ether/α-peptide

27.3 8.6 10.1 13.1 4.7 20.4 11.1 11.5 21.8 17.7

1.9 1.5 5.5 4.6 3.1 0.9 2.3

5.1 0.1 0.1 tr 0.1 tr 4.2

0.9 2.7

tr tr

β-5

β-β

dibenz

50.3 50.8 54.1 54.1 62.3 57.7 52.5 51.7 54.1 53.2

19.2 32.4 30.2 27.2 29.9 20.3 34.1 32.5 22.2 26.2

1.2 1.6 tr 0.9 tr 0.6 tr tr 1.0 0.2

The table shows lignin interunit linkage ratios (as percentage of total α-signal) for DHP and lignin−peptide adducts. In the case of DHP−YGG the DHP−α-peptide shift was degenerate with standard lignin α-O-aryl shifts; thus, the β-ether/α-O-aryl and β-ether/α-peptide quantities were combined. tr, trace (