title: Equilibrium unfolding of Bombyx mori glycyl-tRNA synthetase.
abstract: Unfolding of Bombyx mori glycyl-tRNA synthetase was examined by multiple spectroscopic techniques. Tryptophan fluorescence of wild type enzyme and an N-terminally truncated form (N55) increased at low concentrations of urea or guanidine-HCl followed by a reduction in intensity at intermediate denaturant concentrations; a transition at higher denaturant was detected as decreased fluorescence intensity and a red-shifted emission. Solute quenching of fluorescence indicated that tryptophans become progressively solvent-exposed during unfolding. Wild type enzyme had stronger negative CD bands between 220 and 230 nm than the mutant, indicative of greater alpha-helical content. Urea or guanidine-HCl caused a reduction in ellipticity at 222 nm at low denaturant concentration with the wild type enzyme, a transition that is 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 unfolding and a parallel two state unfolding; the two-state component is absent in the mutant. Changes in spectral properties associated with unfolding were largely reversible after dilution to low denaturant. Unfolding of glycyl-tRNA synthetase is complex with a native state, a native-like monomer, partially unfolded states, and the unfolded state.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Ohio, Block Health Science Building, Toledo, Ohio 43614-5804, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Dignam
authors.forename: X
authors.initials: X
authors.lastname: Qu
authors.forename: J B
authors.initials: JB
authors.lastname: Chaires
mesh_terms: D000818:Animals; D001704:Biopolymers; D012831:Bombyx / Q000201:enzymology*; D002942:Circular Dichroism; D006032:Glycine-tRNA Ligase / Q000737:chemistry*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
chemical_list: D001704:Biopolymers; D014364:Tryptophan; D006032:Glycine-tRNA Ligase