DETERMINATION OF ENDOSPERM PROTEIN SECONDARY STRUCTURE IN HARD WHEAT BREEDING LINES USING SYNCHROTRON INFRARED MICROSPECTROSCOPY AND REVELATION OF SECONDARY STRUCTURAL CHANGES IN PROTEIN FILMS WITH THERMAL PROCESSING

Fourier transform infrared microspectroscopy was used to determine protein secondary structure in hard wheat breeding lines in situ, providing a molecular means to rank endosperm hardness for the selection of wheat cultivars for a specific end-use. Mapping with a single masked spot size diameter of 4.5 µm or confocal 5 µm on beamlines U10B and U2B, respectively, produced spectra from the subaleurone layer within each wheat kernel using the high spatial resolution available with synchrotron infrared microspectroscopy. This procedure was used for the first four crop years. A focal plane array instrument was adapted for use for the remaining two crop years with a slight reduction of spatial resolution. Deconvolution and curve fitting were applied to the amide I region of spectra selected from the interstitial protein between the starch granules, and the relative amount of α-helix to other protein secondary structures was revealed. Over six crop years, the α-helix to β-sheet ratio of experimental wheat varieties were compared to those of released varieties in 143 mapping experiments. The highest measurable value was 2.50 while the lowest was 1.11, a range consistent with hard wheat secondary structure determination found in previous studies (13, 16). The determination of protein secondar