CHARACTERIZATION OF A MOLTEN GLOBULE INTERMEDIATE DURING GDNHCL-INDUCED UNFOLDING OF RTEM BETA-LACTAMASE FROM ESCHERICHIA-COLI

GdnHCl-induced unfolding and reversible folding of beta-lactamase from E. coli have been investigated by measuring enzymatic activity, fluor escence emission and far-UV circular dichroism as indices of the exten t of denaturation. The non-coincidence of far-UV CD and fluorescence d ata and existence of an inflection point clearly suggest the presence of an equilibrium intermediate. The existence of the equilibrium inter mediate at around 1 M is corroborated by its enhanced binding of fluor ophobic probe 1,8-ANS. The intermediate was found to have a compact sh ape as measured by its Stokes radius by size-exclusion chromatography. Furthermore, near-UV CD analysis of this enzymatically inactive inter mediate showed a significantly disrupted tertiary structure with only a minor change in the secondary structure, which is a characteristic o f typical molten globule states. Estimation of the activation energy f rom the kinetics of unfolding of the protein monitored by fluorescence and CD suggests that the intermediate may be separated from the nativ e and the unfolded state by a high activation-energy barrier.