Abstract
Tyrosyl-tRNA synthetase from Bacillus stearothermophilus comprises an N-terminal domain (residues 1-319), which is dimeric and forms tyrosyladenylate, and a C-terminal domain (residues 320-419), which binds the anticodon arm of tRNATyr. The N-terminal domain has the characteristic fold of the class I aminoacyl-tRNA synthetases. The unfolding of the N-terminal domain by urea at 25 degreesC under equilibrium conditions was monitored by its intensities of light emission at 330 and 350 nm, the ratio of these intensities, its ellipticity at 229 nm, and its partition coefficient, in spectrofluorometry, circular dichroism, and size-exclusion chromatography experiments, respectively. These experiments showed the existence of an equilibrium between the native dimeric state of the N-terminal domain, a monomeric intermediate state, and the unfolded state. The intermediate was compact and had secondary structure, and its tryptophan residues were partially buried. These properties of the intermediate and its inability to bind 1-anilino-8-naphthalenesulfonate showed that it was not in a molten globular state. The variation of free energy deltaG(H2O) and its coefficient m of dependence on the concentration of urea were, respectively, 13.8 +/- 0.2 kcal.mol-1 and 0.9 +/- 0.1 kcal.mol-1.M-1 for the dissociation of the native dimer and 13.9 +/- 0.6 kcal.mol-1 and 2.5 +/- 0.1 kcal.mol-1.M-1 for the unfolding of the monomeric intermediate.
Highlights
Tyrosyl-tRNA synthetase from Bacillus stearothermophilus comprises an N-terminal domain, which is dimeric and forms tyrosyladenylate, and a C-terminal domain, which binds the anticodon arm of tRNATyr
Tyrosine is first activated with ATP to form tyrosyladenylate, and this intermediate is attacked by tRNATyr to form tyrosyl-tRNATyr and AMP
The difference in fluorescence intensity between the native and unfolded states of TyrRS(⌬1) was maximal at an emission wavelength of 330 nm. This difference was larger when the excitation wavelength was equal to 278 nm than when it was 295 nm, as expected since both tyrosine and tryptophan are excited at 278 nm, whereas only tryptophan is excited at 295 nm
Summary
Tyrosyl-tRNA synthetase from Bacillus stearothermophilus comprises an N-terminal domain (residues 1–319), which is dimeric and forms tyrosyladenylate, and a C-terminal domain (residues 320 – 419), which binds the anticodon arm of tRNATyr. The unfolding of the N-terminal domain by urea at 25 °C under equilibrium conditions was monitored by its intensities of light emission at 330 and 350 nm, the ratio of these intensities, its ellipticity at 229 nm, and its partition coefficient, in spectrofluorometry, circular dichroism, and size-exclusion chromatography experiments, respectively These experiments showed the existence of an equilibrium between the native dimeric state of the N-terminal domain, a monomeric intermediate state, and the unfolded state. We have shown that the isolated form of the C-terminal domain, TyrRS(⌬3), has secondary structure, is compact, and unfolds through a cooperative transition [15]. We detected a monomeric intermediate between the native dimeric state of the N-terminal domain and its unfolded state and quantified the variation of free energy and its dependence on the concentration of urea for each of the corresponding conformational transitions. The quantitative thermodynamic analysis developed in this work should be applicable to other dimeric or oligomeric proteins
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