Abstract
The present work aimed to characterize the molecular relationships between structure and function of the seed storage protein β-vignin, the vicilin storage protein of cowpea (Vigna unguiculata, l. Walp) seeds. The molecular characterization of β-vignin was carried out firstly by assessing its thermal stability, under different conditions of pH and ionic strength, by thermal shift assay (TSA) using SYPRO Orange fluorescent dye. Secondly, its aggregation propensity was evaluated using a combination of chromatographic and electrophoretic techniques. Two forms of β-vignin were considered: the native form purified from mature quiescent seeds, and a stable breakdown intermediate of 27 kDa produced while seeds germinate. TSA is a useful tool for determining and following over time the structural changes that occur to the protein during germination. The main result was the molecular characterization of the 27 kDa intermediate breakdown polypeptide, which, to the best of our knowledge, has never been described before. β-vignin seems to retain its trimeric conformation despite the evident degradation of its polypeptides.
Highlights
Proteins account for about 22–28% of dry cowpea
The present work investigated the stability and aggregative properties of both native β-vignin and proteolytic products of β-vignin transiently accumulated during seed germination, at different pH and ionic strength conditions
thermal shift assay (TSA) has proved to be a useful tool for determining and following over time the structural changes that occur during germination
Summary
Proteins account for about 22–28% of dry cowpea Β-vignin is the vicilin protein of cowpea seeds [3]. It is present in two main isoforms with molecular weights of 60 and 56 kDa, differently glycosylated and without disulfide bridges [6]. SPs are traditionally considered the nitrogen reserve that support seedling growth during the first steps of germination [7] This role has been recently ruled out and new findings indicate that several biological activities become manifest upon proteolytic breakdown. The enzymes involved, and the Cowpea β-vignin molecular properties, up to now, have been investigated by few intermediates generated, are still largely unknown. We undertook the present study as partand of our research activity aimed to characterize the molecular relationships between structure function of seed storage proteins [14–. Mature quiescent seeds and the breakdown breakdown intermediates produced while seeds intermediates produced while seeds germinate
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