Cruciferin-enriched fraction
Many of the FT-IR spectral features of the camelina cruciferin-enriched fraction (Figure 6) were similar those from A. thaliana(Withana-Gamage et al., 2013) and B. napus (Perera et al., 2016) with subtle differences observed for the -PO3 (970 cm-1), C-O-P (1070 cm-1) and -P=O (1170 cm-1) functional groups. The estimated values for secondary structure components based on deconvolusion of the amide I band 1600-1690 cm-1 (Figure 6) were 43% ± 0.9% β-sheets, 10.1% ± 0.3% α-helix, 19.8% ± 0.8% β-turns and 3.9% ± 0.6% random structure. Comparable levels of β-sheet structure were reported for B. napus (45.6%; Perera et al., 2016) and A. thaliana (~44%; Withana-Gamage et al., 2013). Camelina cruciferin structure exhibited acidic pH-induced, secondary structure changes as observed in the far-UV CD spectra (Figure 7A). The values for molecular ellipticity (θ) changed and α-helix content significantly increased (11.6% ± 0.3%) at pH 3, but not at pH 10 (Figure 7A and Table 4), compared to that at pH 7 (2.9% ± 0.2%) (Table 4).Brassica napus cruciferin also exhibits unfolding at acidic pH (Perera et al., 2016).
An increase in protein surface hydrophobicity values (S0) at pH 3 (7393.1 ± 32) compared to that at pH 7 (557.8 ± 2.4) (Table 4) indicates exposure of hydrophobic residues that had been buried (Korte and Herrmann, 1994). Together with the changes in intrinsic fluorescence of the Trp residue, the observed decrease in the maximum fluorescence intensity (Fmax), red shift in the maximum emission wavelength (λmax), and a higher ratio of fluorescence intensity (F) at 350 nm and 330 nm at pH 3 compared to neutral pH (F350/F330 =1.13 at pH 3 and 0.71 at pH 7) (Figure 7 C and D) all support acidic pH-induced unfolding of camelina cruciferin tertiary structure. Similar behaviour has been reported in acidic environments for cruciferins from other Crucifer species (Perera et al., 2016; Withana-Gamage, 2013).
Camelina cruciferin showed a significantly (p<0.05) higher maximum denaturation temperature at pH 10 (Tm=83.1°C) than at pH 7 (Tm=80.6°C), but similar thermal energies (enthalpy, -ΔH) were required for denaturation (Table 4) indicating high thermal stability of the structure. The absence of a thermal transition peak at pH 3 (Table 4) further supported the structural unfolding indicated in other analyses. The intrinsic fluorescence data (Fmax, λmax and F350/F330) (Figure 8) obtained for protein heated above 83°C (or the denaturation temperatures) at pH 7 and 10 (Figures 8 B and C) were similar to the values at pH 3 at ambient temperature (22°C). This confirms the existence of unfolded/denatured structure due to heat or acidic pH. The predominant native quaternary structure for camelina cruciferin may be a mixture of trimers and hexamers (as indicated from native PAGE) below 80°C at both pH 7 and 10 (Figures 8B and C). The predominance of unfolded structure was evident as the temperature increased above 83°C, similar to what was observed for B. napus cruciferin (Perera et al., 2016). The higher values for denaturation temperature (95.5°C, ΔH=~14 J g-1 for cold-pressed meal and 93.3°C, ΔH=9 J g-1 for hot-pressed meal) reported for camelina proteins (Boyle et al., 2018) may be related to other associated proteins/components in the tested material, as indicated from the ~79-82% purity based on total N, or to structural alterations at highly alkaline pH (pH 12, alkali-induced unfolding) during extraction and (NH4)2SO4 precipitation.
In B. napus cruciferin, acid-induced dissociation is considered reversible, whereas heat-induced denaturation causes irreversible changes (Wanasundara, 2011). At pH 3, B. napus cruciferin assumes a molten globule structure (Perera et al., 2016; Schwenke and Linow, 1982), a partially-folded conformation of a globular protein with near-native compactness, significant secondary structure, insignificant tertiary structure and a substantial amount of solvent-exposed hydrophobic residues compared to its native state. The behaviour exhibited in the structural features and their changes during pH destabilization indicate that camelina cruciferin also assumes a molten globule state under acidic conditions that is somewhat reversible.