Protein structure analyses
Fourier Transform-Infrared (FTIR) spectroscopy : FT-IR
spectroscopy was conducted using an Agilent Cary 630 ATR-FTIR analyzer
(Agilent Technologies Inc.) according to Perera et al. (2016). Secondary
structure details of proteins were obtained by Fourier
self-deconvolution (FSD) of the amide I region (1610–1700 nm) from the
spectra using Agilent Resolution Pro version 5.2.0 with the following
parameters: resolution enhancement factor (K) = 2.5, full width at half
height = 14 cm-1 and apodization filter = Bessel for
cruciferin, whereas K = 2.8 and full width at half height = 18
cm-1 were used for napin.
Circular Dichroism (CD) : Far-UV spectra of proteins [1 mg/mL
prepared in 10 mM sodium citrate buffer (pH 3), 10 mM sodium phosphate
buffer (pH 7) or 10 mM ammonium chloride/ammonia buffer (pH 10)] were
acquired at 25°C using a PiStar-180 spectrometer (Applied Photophysics
Ltd.) as described by Perera et al. (2016) and Withana-Gamage et al.
(2013). Molar ellipticity of proteins was calculated with background
correction using the CDNN 2.1 software package (Applied Photophysics
Ltd.).
Intrinsic fluorescence : The fluorescence emission spectra of
protein slurries (5%, w:v, cruciferin and 10%, w:v, napin prepared at
pH 3, 7 and 10 in the buffer systems noted above) were monitored for the
tryptophan residue (excitation at 280 nm and emission from 290-450 nm)
in the temperature range of 22-93°C using a fluorescence
spectrophotometer (Agilent Eclipse, Model G9800A, Agilent Technologies
Inc.).
Surface hydrophobicity with anionic fluorescent probing : Binding
of 1-anilino-8-napthalensulfonate (ANS, an anionic fluorescence probe)
with hydrophobic regions on the surface of the protein in solution
(0.05–0.25 mg/mL at pH 3, 7 and 10) was measured. The fluorescence
probe was excited at 390 nm and the emission monitored at 470 nm and 500
nm for cruciferin and napin, respectively, using a Cary eclipse
fluorescence spectrophotometer (Agilent Technologies Inc.). Surface
Hydrophobicity Index (S0) was calculated as described in
Perera et al. (2016) and Withana-Gamage et al. (2013).
Thermal stability assessment using differential scanning
calorimetry (DSC) : Protein slurries (5%, w:v, cruciferin and 10%,
w:v, napin) prepared at pH 3, 7 and 10 using the buffer systems noted
above were used for DSC analysis. Samples were placed into aluminum
pans, hermetically sealed with a Tzero™ press (TA Instruments) and
temperature-induced structural changes recorded from 30°C to 130°C under
constant nitrogen purging using a TA Q2000 differential scanning
calorimeter (TA Instruments). An empty, hermetically sealed pan was used
as a reference and the thermograms analysed using TA universal analysis
2000 software (TA Instruments).