Equilibrium unfolding and refolding of black gram (Vigna mungo) phaseolin

Abstract

Equilibrium unfolding of black gram phaseolin (BGP) using guanidine hydrochloride and urea as denaturants, thermal unfolding, and refolding were studied using several spectroscopic probes and electrophoretic techniques. UV absorbance and intrinsic fluorescence revealed a three-stage unfolding process, while CD spectroscopy, extrinsic fluorescence probe ANS, and thermal denaturation followed a two-state unfolding transition. The ?-sheet was more resistant to chemical denaturation than the ?-helical regions of BGP. BGP and an analogous navy bean phaseolin (NBP) could revert back to their protomeric conformations from their partially unfolded states in 2 M denaturant concentrations, while only NBP could regain its native conformation from the completely unfolded state. BGP completely unfolded in urea and upon thermal denaturation could never regain its native structure. These differences in the refolding of two otherwise homologous proteins from their completely unfolded states were discussed in terms of why BGP is a preferred legume in mixed legume cereal fermentations. Practical applications: The major storage proteins of black gram and dry beans exhibited significant differences in their unfolding refolding behavior. Unlike the dry bean phaseolin that refolds back to its native structure quite quickly, black gram phaseolin was able to maintain its unfolded state; thereby allowing the formation of soft, porous, and spongy network in several popular fermented and steamed products on the Indian subcontinent. This may explain the popularity of black gram in such mixed cereal legume fermentations. Black gram is thus ideally suited for legume-fortified food products where such texture is desired. Furthermore, understanding of the folding unfolding behavior of major storage proteins of legume will enhance our ability to genetically modify these proteins with sulfur-rich peptides to improve their nutritional quality by identifying the key structural motifs in these proteins that are essential and vital to the proper folding of the protein in situ in genetically modified seeds. 2018 Wiley Periodicals, Inc.