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 tRNA Tyr . 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°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. 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 ⌬G(H 2 O) and its coefficient m of dependence on the concentration of urea were, respectively, 13.8 ؎ 0.2 kcal⅐mol ؊1 and 0.9 ؎ 0.1 kcal⅐mol ؊1 ⅐M ؊1 for the dissociation of the native dimer and 13.9 ؎ 0.6 kcal⅐mol ؊1 and 2.5 ؎ 0.1 kcal⅐mol ؊1 ⅐M ؊1 for the unfolding of the monomeric intermediate.

A dense cluster of eight residues was identi®ed at the crossing of two a-helices in tyrosyl-tRNA synthetase (TyrRS) from the thermophile Bacillus stearothermophilus. Its mechanism of evolution was characterized. Four residues of this cluster are not conserved in TyrRS from the mesophile Escherichia coli. The corresponding mutations were constructed in TyrRS(Á1), a derivative of TyrRS from B. stearothermophilus in which the anticodon binding domain is deleted. Mutations I52L (i.e. Ile52 into Leu), M55L and L105V did not affect the activity of TyrRS(Á1) in the pyrophosphate exchange reaction whereas T51P increased it. The kinetic stabilities of TyrRS(Á1) and its mutant derivatives at 68.5 C were determined from experiments of irreversible thermal precipitation. They were in the order L105V < I52L < T51P < Wild Type 4 M55L; mutation I52L partially compensated L105V in these experiments whereas M55L was coupled neither to I52L nor to L105V. Mutations I52L and L105V affected the stability of the dimeric TyrRS(Á1) at different steps of its unfolding by urea, monitored under equilibrium conditions by spectro¯uorometry or size exclusion chromatography. I52L destabilized the association between the subunits even though residue Ile52 is more than 20 A Ê away from the subunit interface. L105V destabilized the monomeric intermediate of unfolding. The two mutational pathways, going from the wildtype TyrRS(Á1) to the I52L-L105V double mutant through each of the single mutants were not equivalent for the stability of the monomeric intermediate and for the total stability of the dimer. One pathway contained two neutral steps whereas the other pathway contained a destabilizing step followed by a stabilizing step. Mutation I52L allowed L105V along the ®rst pathway and compensated it along the second pathway. Thus, the effects of I52L and L105V on stability depended on the structural context. The gain in activity due to T51P was at the expense of a slight destabilization.