Thermal Shift Assay as a Tool to Evaluate the Release of Breakdown Peptides from Cowpea β-Vignin during Seed Germination

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 ( , l. Walp) seeds. The molecular characterization of β-vignin was carried out firstly by assessing its thermal stability, und...

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Bibliographic Details
Published in:Molecules (Basel, Switzerland) Switzerland), 2022-01, Vol.27 (1), p.277
Main Authors: De Benedetti, Stefano, Leogrande, Camilla, Castagna, Francesco, Heinzl, Giuditta C, Pasquali, Matias, Heinzl, Alessandro L, Lupi, Daniela, Scarafoni, Alessio
Format: Article
Language:English
Subjects:
Aggregates
bioactivity
Breakdown
Chromatography
Cowpeas
Electrophoresis
Enzymes
Evaluation
Fluorescent dyes
Fluorescent indicators
Germination
Ionic strength
Molecular structure
Peptides
Peptides - metabolism
Polypeptides
Proteins
proteolysis
Seed germination
seed storage proteins
Seed Storage Proteins - metabolism
Seeds
Seeds - metabolism
Stability analysis
Structure-function relationships
Thermal stability
Vigna - metabolism
Vigna unguiculata
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Summary: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 ( , 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.
ISSN:1420-3049
1420-3049
DOI:10.3390/molecules27010277