Equilibrium unfolding of Bombyx mori glycyl-tRNA synthetase

Unfolding of Bombyx mori glycyl-tRNA synthetase was examined by multiple sp ectroscopic techniques. Tryptophan fluorescence of wild type enzyme and an N-terminally truncated form (N55) increased at low concentrations of urea o r guanidine-HCl followed by a reduction in intensity at intermediate denatu rant concentrations; a transition at higher denaturant was detected as decr eased fluorescence intensity and a red-shifted emission. Solute quenching o f fluorescence indicated that tryptophans become progressively solvent-expo sed during unfolding. mild type enzyme had stronger negative CD bands betwe en 220 and 230 Bm than the mutant, indicative of greater alpha -helical con tent. Urea or guanidine-HCl caused a reduction in ellipticity at 222 nm at low denaturant concentration with the wild type enzyme, a transition that i s absent in the mutant; both enzymes exhibited a cooperative transition at higher denaturant concentrations. Both enzymes dissociate to monomers in 1. 5 M urea. Unfolding of wild type enzyme is described by a multistate unfold ing and a parallel two state unfolding, the two-state component is absent i n the mutant. Changes in spectral properties associated with unfolding were largely reversible after dilution to low denaturant. Unfolding of glycyl-t RNA synthetase is complex with a native state, a native-like monomer, parti ally unfolded states, and the unfolded state.