Key points are not available for this paper at this time.
β-vignin is the main storage protein in cowpea (Vigna unguiculata, Walp) seeds. Similar to other vicilins, differently glycosylated isoforms organize in trimers whose oligomerization is influenced by salt concentration and pH. It was previously observed that two polypeptides, resistant to further proteolysis, are generated and persist throughout the germination process. This study aimed to characterize the structural features of partially degraded, yet structured, cleaved β-vignin using a combination of biochemical and biophysical techniques. The cleavage site is present only in the non-glycosylated isoform, located between the two cupin domains. Thermal Shift Assay (TSA), Differential Scanning Calorimetry (DSC) and thermodynamic analysis of β-vignin in its native form highlighted the complex stability scenario, in line with the structural evidence that accounts for two isoforms and the monomer-to-trimer equilibrium. Furthermore, DSC data suggest that the protein's tertiary and quaternary structure is resilient to the structural modifications resulting from enzymatic cleavage. The tertiary conformation is "released" from some constraints of the primary structure, producing an even more compact protein. In addition, by exploiting the pH-dependent dissociation equilibrium of β-vignin, we investigated how glycosylated and non-glycosylated β-vignin isoforms combine to form the trimer. This study, revealing that the stable breakdown intermediate maintains a higher-order structure, provides novel insights into the current understanding of seed storage protein dynamics during germination. The molecular characterisation of the stable breakdown products is instrumental in further investigating their functional roles during germination, such as insecticidal or antifungal properties, and/or as mediators of seed adaptation to environmental conditions.
Periccioli et al. (Wed,) studied this question.