Characterization of a urea induced molten globule intermediate state of glutaminyl-tRNA synthetase from Escherichia coli.

The urea-induced unfolding of glutaminyl-tRNA synthetase, a multidomain protein, has been studied by equilibrium and kinetic methods, using chemical modification, fluorescence, and CD spectroscopy. The far-UV CD, fluorescence, and sulfhydryl reactivity clearly demonstrated the existence of a stable intermediate state at around 2 M urea. The intermediate showed higher binding of 1-anilino-8-naphthalenesulfonic acid. Furthermore, near-UV CD study of the intermediate showed significantly disrupted tertiary structure with only a small change in the secondary structure, which is a characteristic of molten globule states. The activation energies (delta G++) calculated from unfolding kinetics monitored by CD and fluorescence suggest that the intermediate state may be separated from the native and the unfolded state by high activation energy barriers.