Dynamics of compact denatured states of glutaminyl-tRNA synthetase probed by bis-ANS binding kinetics

Bis-ANS binds to native glutaminyl-tRNA synthetase (GlnRS) with a fast and a slow phase. The rate constant of the slow phase is independent of bis-ANS concentration suggesting a slow conformational change in the pathway of bi s-ANS binding. Aging of GlnRS causes a large decrease of the slow phase amp litude with concomitant increase of the fast phase amplitude. Several other large, multi-domain proteins show similar patterns upon aging. The near UV -CD spectra of the native and the aged GlnRS remain similar. Significant ch anges in far UV-CD, acrylamide quenching and sulfhydryl reactivity, are see n upon aging, suggesting disruptions in native interactions. Refolding of G lnRS from the urea-denatured state rapidly produces a state that is very si milar to the equilibrium molten globule state. Bis-ANS binds to the molten globule state with kinetics similar to that of the aged state and unlike th at of the native state. This suggests that the slow binding phase of bis-AN S, seen in native proteins, originate from relatively high energy barriers between the native and the more open states. Thus bis-ANS can be used as a powerful probe for large amplitude, low-frequency motions of proteins. (C) 2000 Elsevier Science B.V. All rights reserved.