Equilibrium Unfolding and Refolding of Black Gram (vignamungo) Phaseolin

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 beta-sheet was more resistant to chemical denaturation than the a-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.