In our previous study26 of C. cryptica using conventional 1D and 2D ssNMR spectroscopy, we observed that a high amount of proteins is associated to the biosilica and we detected two different chitin conformations. In the following, we used DNP-supported NMR experiments to further enhance ssNMR signal intensities. As mentioned above, DNP relies on spin polarization transfer from electron spins to nuclei. In our experiments, samples were impregnated with a “DNP juice” (see methods) containing the biradical AMUPol32. First, we conducted one-dimensional 13C and 15N detected CP experiments (Fig. 1) under DNP conditions and, as in our previous study3, defined the DNP enhancement factor by the ratio of signal intensities with and without microwave irradiation. In 13C CP experiments, we observed enhancement factors that (except for carbohydrates) ranged between 18 and 26 (Fig. 1, left). In the 15N CP measurement, we observed an enhancement of 26 for both amide and amine 15N signals (Fig. 1, right). As we have discussed elsewhere26, C. cryptica contains a massive organic matrix and produces extracellular chitin fibrils of ca. 50 nm thickness33–34 (see also Figure S1).
To further investigate molecule-specific DNP enhancements, we performed a two-dimensional DNP-enhanced PDSD (Proton-Driven Spin Diffusion) experiment (Fig. 2). We observed similar 13C-13C correlations as in the standard PDSD experiment26, however with considerably higher signal intensities. This enabled the detection of new signals which are not observable in the conventional experiment. In particular, two spin systems with several correlations are observable: One spin system shows correlations from 92 ppm to 81 ppm, 75 ppm, and 72 ppm. The other system shows correlations from 95 ppm to 81 ppm, 75 ppm, 73 ppm, and 70 ppm. (Fig. 2, framed in blue). These correlations are characteristic for carbohydrates, where the C1 is not connected. These unconnected anomeric carbon atoms characteristically give rise to peaks between 90 and 100 ppm. In this case both spin systems also contain a shift higher than 80 which indicates a substitution on the carbocycle35 by e.g. another sugar. This points towards the presence of reducing ends of polysaccharides36. A monosaccharide analysis which provides information about the total carbohydrate composition revealed glucose and mannose as main monosaccharides. Xylose, ribose, fucose, galactose and glucosamine are also present in significant concentrations26. However, a differentiation between monosaccharides and monosaccharide units from polysaccharides is not possible due to the performed hydrolysis step.
Moreover, the determination of DNP enhancement factors for different signals, which are characteristic for different organic compounds, can help to probe the supramolecular architecture of the biosilica3. To distinguish DNP enhancements of protein and carbohydrate signals, we analyzed different 1D slices of the 2D spin diffusion experiment (Fig. 2). For the protein signals, we examined a 1D slice at 53 ppm which is typical for protein backbone Cα resonances and observed a strong enhancement factor of 26 in line with our 1D 15N CP signals (Fig. 1). Thus, the proteins should be located at the solvent-accessible surface of the biosilica, where the radical solution (AMUPol) can polarize nuclei located in the biomolecules. In contrast, for the carbohydrate region, e.g. the signals at 105 ppm, we determined significantly lower enhancement factors between 5–8 which is close to the 13C enhancements seen in 1D CP MAS for carbohydrates (Fig. 1). Note that in the latter case, a precise determination of the DNP enhancements for carbohydrates may be complicated due to the overlapping MAS sidebands from other carbon species.
As discussed elsewhere3, 19, 37, the DNP enhancement in complex biomolecules depends on the internuclear distances between hydrogens responsible for the polarization transfer via proton-driven spin diffusion and the macromolecular layer size. In addition, the DNP enhancement for biomolecules close to the radical, may itself be modulated by the details of the local electron-nucleus geometry. This geometry determines the spin-diffusion barrier38, influences the DNP magnetic field dependence of the DNP effect39 and leads to paramagnetic line broadening14, 40 within a sphere of about 1 nm around the DNP radical14. We expect this effect to be particularly strong for biosilica such as from S. turris which are characterized by small protein/organic surface layers3. Indeed, DNP signal enhancements for polyssacharides and polyamines amounted to about 40% to the protein signal enhancements for S. turris3. In contrast, the organic layer for C. cryptica is significantly larger26 which results in almost uniform DNP enhancements in 1D 13C CP MAS data and is possibly the reason for the larger (relative) decrease in DNP enhancement from 26 to 5–8 for carbohydrates.
Note that the increased line broadening often observed at low-temperature DNP conditions14 prevents the differentiation between the α-like chitin- and β-chitin signals as it was possible without DNP and at ambient sample temperatures26. On the other hand and as mentioned earlier, C. cryptica contains a massive organic matrix and produces extracellular chitin fibrils of ca. 50 nm thickness33–34. Hence, a more detailed analysis employing a classical spin-diffusion approximation to correlate relative DNP enhancements with biosilica layer thickness as done earlier for labeled diatom biosilica of S. turris 3 was not attempted here. Instead we can draw the following general conclusions. Firstly, we would expect from previous theoretical work from our laboratory37 only minor changes for the DNP enhancement for chitin embedded in the extracellular chitin fibrils compared to the protein layer. On the other hand, fluorescence spectroscopy41 has shown that chitin in C. cryptica biosilica can also be directly associated to the siliceous cell walls. For this species, that is potential surrounded by low density of hydrogens that limit the spin-diffusion process, low DNP enhancements as found in Fig. 2 may be possible. Since ssNMR detects the entire ensembles of chitin moieties in our sample, the latter species may hence represent a prominent fraction in our C. cryptica preparations.
To further investigate the carbohydrate association to the siliceous cell walls, we conducted similar experiments on isotope-enriched T. pseudonana biosilica (Fig. 3) for which the presence of a chitin-based meshwork is already known11. Again, we analyzed 1D slices typical for protein and carbohydrate signals and observed a strong enhancement factor of 32 for the protein Cα signals (signal at 53 ppm). In line with our earlier observations, the proteins are thus predominately located at the solvent-accessible surface of the biosilica, where the radical solution (AMUPol) can polarize nuclei located in the biomolecules. Remarkably, for the carbohydrate region, e.g. the signals resonating at 105 ppm, a lower enhancement compared to the protein signals is again observed. However, the relative enhancement of about 15 is significantly larger than in the case of C. cryptica biosilica (amounting to 5–8). These findings would be consistent with the notion that the organic matrix of T. pseudonana biosilica is less pronounced than in the case of C. cryptica 26 but larger than for S. turris. Such an interpretation would be consistent with previous findings42.
Moreover, our DNP experiments revealed the presence of additional carbohydrate species as seen before for C. cryptica (Fig. 2). Interestingly, one of the observed new sugar spinsystems exhibits a correlation of the anomeric carbon to a signal around 165 ppm (Figure S2). Sp2-configured carbon atoms of nitrogen containing functional groups including imines43 and guanidiniums44 as well as amide carbonyls give rise to peaks in this region. This correlation could hence indicate a cross linking between a carbohydrate C1 and an amino acid such as the guanidinium containing arginine, which has rarely been described as part of N-glycans. Another possibility would be the presence of citrulline as its carboxamide gives rise to resonances at around 164 ppm and it has been described as being linked to polysaccharides before45. This link could represent the interface of the sugarlayers with the residual organic matrix.
Finally, we used DNP-supported 15N-13C correlation experiments to further investigate proteins as well as the presence of LCPA in our preparations. Again, DNP greatly facilitated such experiments because of the increased spectroscopic sensitivity, thereby reducing measurement times. To study nitrogen containing 13C moieties, we performed 15N filtered experiments30–31. Figure 4 (red spectrum) shows an 15N-13C correlation spectrum where magnetization is first transferred from 1H to the 15N labelled amines of the LCPAs followed by a 15N-13C CP transfer to nearby 13C carbons (typically separated by one chemical bond in fully labelled proteins31). The introduction of a spin diffusion (mixing) step between carbons allowed us to study more distant 13C atoms including polyamines (blue spectrum). Figure 4 shows the 15N-13C correlations of the LCPA region, exclusively. The former discussed NMR spectra show the presence of LCPAs within the biosilica sample. Carbon signals between 30–70 ppm correlate with nitrogen signals between 30–60 ppm. Signals between 20–60 ppm in the nitrogen dimension correspond to the primary, secondary and tertiary amines of LCPAs as well as lysine side chains. These correlate with carbon signals between 45–70 ppm characteristic for carbon atoms that are connected to amines. Due to the expected manifold of LCPAs in the sample, we detected various intense correlation signals. In the NCC experiment with a mixing time of 50 ms, correlations between carbon atoms with longer distances to the amine-nitrogen can be observed. Thus, an increasing number of correlations occurs, e.g., correlations to alkyl groups at lower 13C chemical shift between 15–30 ppm. Interestingly, we even observed a correlation to carbonyl-carbons at 174 ppm. This signal occurs probably due to relay transfer between close-by 13C labeled atoms to a carbonyl atom as usually seen in fully labeled protein samples under MAS conditions (see e.g. Ref.46) Such a correlation between the amine of a LCPA and a carbonyl group was not observed for other diatom biosilica, including diatom biosilica from S. turris3. However, it is well known from the model organism T. pseudonana11 that LCPAs can be covalently attached to lysine residues or posttranslational modified hydroxylysine residues that may give rise to the correlation seen at 174 ppm. Additionally, the chemical shift of this carbonyl group is more typical for carbonyl groups of proteins. The above discussed chitin should give rise to a signal at a higher chemical shift of ca. 176 ppm. Thus, proximity between amine nitrogens and protein carbonyl groups is more likely here.