The thermal denaturation behaviour of glycinin solutions has been studied in situ as a function of ionic strength using various spectroscopic methods. Changes in secondary structure occurred at temperatures above 60 °C, well before the onset of gelation. Even after heating to 95 °C, much of the native ß-sheet structure of glycinin was retained, as indicated by the amide I peak maximum at 1635 cm-1 in the Fourier transformed infrared (FT-IR) spectrum. This was accompanied by an increase in the 1625 cm-1 band, indicative of the formation of intermolecular ß-sheet associated with protein aggregation. Nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of highly mobile regions in glycinin comprising predominantly of Gln and Glu residues, corresponding to mobile regions previously identified by crystallographic studies. There was also evidence of a hydrogen-bonded structure within this mobile region, which may correspond to an a-helical region from Pro256 to (or just before) Pro269 in proglycinin. This structure disappeared at 95 °C, when heat-set gel formation occurred, as indicated by a sudden broadening and weakening of the NMR signal. Otherwise the NMR spectrum changed little during heating, emphasising the remarkable thermal stability of glycinin. It is proposed that during heating the core ß-barrel structure remains intact, but that the interface between the ß-domains melts, revealing hydrophobic faces which may then form new structures in a gel-network. As Cys45, which forms the disulfide with Cys12 linking the acidic and basic polypeptides, is found in this interface, such a rearrangement of the individual ß-domains could be accompanied by cleavage of this disulfide bond, as is observed experimentally. Such information contributes to our understanding the aggregative behaviour of proteins, and hence develops knowledge-based strategies for controlling and manipulating it.
|Number of pages||10|
|Journal||Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics|
|Publication status||Published - 2003|