Unfolding and aggregation-associated changes in the secondary structure of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride as monitored by FTIR

Abstract

The secondary structure of native D-glyceraldehyde-3-phosphate dehydrogenase was compared with its partially folded intermediate and aggregated states obtained during guanidine hydrochloride (GdnHCl) denaturation using transmission Fourier transform infrared (FTIR) and micro-FTIR measurements. The changes in the secondary structures indicated a partially folded intermediate formed in 0.1M GdnHCl solution without visible aggregation. Increasing the GdnHCl concentration resulted in aggregation of the enzyme along with changes in the secondary structure. Although similar relative amounts of the secondary structure were found in the aggregated and native states of the enzyme, the temporal variation of the secondary structure revealed a difference in the β-sheet structure between the aggregated and native states, suggesting that the aggregation resulted from further unfolding of the enzyme. In addition, FTIR data suggest that such aggregates are most likely mediated by specific intermolecular interactions and that the predominant driving force involved in aggregation may be a hydrophobic interaction between exposed surfaces of partially folded intermediates. © 1997 John Wiley & Sons, Inc. Biospect 3: 121–129, 1997