CHARACTERIZATION OF A UREA INDUCED MOLTEN GLOBULE INTERMEDIATE STATE OF GLUTAMINYL-TRANSFER-RNA SYNTHETASE FROM ESCHERICHIA-COLI

The urea-induced unfolding of glutaminyl-tRNA synthetase, a multidomai n protein, has been studied by equilibrium and kinetic methods, using chemical modification, fluorescence, and CD spectroscopy. The far-UV C D, fluorescence, and sulfhydryl reactivity clearly demonstrated the ex istence of a stable intermediate state at around 2 M urea. The interme diate showed higher binding of 1-anilino-8-naphthalenesulfonic acid. F urthermore, 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 ac tivation energies (Delta G(double dagger) calculated from unfolding ki netics 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.