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 ␣-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.Aminoacyl-tRNA synthetases are a structurally diverse group of enzymes divided into two classes based on the topography of their adenylate binding sites and their modes of tRNA binding (1-3). The active sites of class I enzymes have a classic Rossman fold formed from two ␤-␣-␤ elements resulting in a structure of four parallel beta strands. The active sites of class II enzymes consist of a unique fold formed from an antiparallel seven-stranded sheet element first identified in seryl-tRNA synthetase (4). A structure similar to the active site of class II enzymes exists in the biotin synthetase/repressor protein (5) and the asparagine synthetase (6), both of which have an adenylate intermediate on their reaction pathways. The two aminoacyl-tRNA synthetase classes have different modes of nucleotide binding and approach tRNA from opposite sides of the polynucleotide. The differences in primary structure and modes of ligand binding suggest that the two classes arose independently (7-9).Glycyl-tRNA synthetases are unusual in that there are two distinct enzyme types that are not closely related. They have developed modes of tRNA recognition that differ with respect to the discriminator base and other base pairs in the stem (10, 11). The type exemplified by the Escherichia coli enzyme is found in Gram-negative and Gram-positive bacteria, whereas a second enzyme is found in other eubacteria such as Mycobacteria sp. and Mycoplasma sp., organisms classified as thermotoga (e.g. Thermus thermophilus), the archaea (e.g. Methanococcus jannashii), and in all eukaryotic organisms. Examination of the crystal structure of T. thermophilus glycyl-tRNA synthetase reveale...

Investigation of the unfolding pathway of Bacillus thuringiensis Cyt2Aa2 toxin reveals an unfolding intermediate

“…1). The shifting of max to a longer wavelength was similar to other reported unfolding proteins such as concanavalin A, methanol dehydrogenase, and glycyl-tRNA synthetase, indicating a conformational change of tryptophan residues from an apolar to a polar environment (Wang et al, 2000;Dignam et al, 2001;Chatterjee and Mandal, 2003). With a series of intensity ratio between 330 and 350 nm representing native and unfolded conformations, an unfolding curve was then established as a function of the denaturant (Fig.…”

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Role of the NC-Loop in Catalytic Activity and Stability in Lipase from Fervidobacterium changbaicum

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