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

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.

Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes

“…It is worth mentioning that fluorescence-based methods can also be used to investigate protein–ligand interaction (PLI) and protein–protein interaction (PPI) because the presence of an interacting ligand or protein can alter the stability of the protein of interest. − Although DSF has been widely used to study protein stability and PLI, − ,− it requires micromolar protein concentration, , potentially promoting protein aggregation and leading to high background signals in the presence of native proteins.…”