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.