Dimeric Tyrosyl-tRNA Synthetase from Bacillus stearothermophilus Unfolds through a Monomeric Intermediate

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 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.

Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization 1 1Edited by A. R. Fersht

“…This prerequisite is well established for dimeric proteins that unfold without intermediate between the native and unfolded states under equilibrium conditions (Neet & Timm, 1994). In contrast, only a handful of papers have reported quantitative analyses of the unfolding equilibria for dimeric proteins that unfold through a dimeric or a monomeric intermediate (Clark et al, 1993;Cheng et al, 1993;Eftink et al, 1994;Grimsley et al, 1997;Park & Bedouelle, 1998). To our knowledge, no detailed study has yet been published on the variations of stability induced by mutations in these types of proteins.…”

“…We have applied them to tyrosyl-tRNA synthetase from Bacillus stearothermophilus (Bst-TyrRS) and established that its unfolding by urea involves a dissociation of the dimer followed by the unfolding of the monomer (Park & Bedouelle, 1998). The quality of the data that we obtained for the wild-type BstTyrRS, suggested to us that it should be possible to quantify the effects of mutations on its stability.…”

“…We have determined conditions in which its thermodynamic equilibrium of unfolding is reached and found that its unfolding is reversible under these conditions (GuezIvanier & Bedouelle, 1996). We have shown that TyrRS(Á1) unfolds through a monomeric state, intermediate between the native dimeric state and the unfolded state, according to the mechanism N 2 @A 2I @A 2U (Park & Bedouelle, 1998). In particular, we have monitored the unfolding of TyrRS(Á1) by its intensity of¯uorescence.…”

“…Because Thr51 appeared peripheral to the cluster, we restricted our analysis of the multiple mutations to those involving residues Ile52, Met55 and Leu105. We used the truncated enzyme Bst-TyrRS(Á1) in this work rather than the full-length Bst-TyrRS, because its unfolding can be analysed quantitatively and its crystal structure is fully known (Park & Bedouelle, 1998;Brick et al, 1989). The changes of residues were constructed by site-directed mutagenesis of phage M13-BY(Á1).…”

“…In particular, we have monitored the unfolding of TyrRS(Á1) by its intensity of¯uorescence. From the unfolding pro®les thus obtained, we have quanti®ed the following thermodynamic parameters: ÁG 1 (H 2 O), the free energy for the dissociation of the dimer subunits; ÁG 2 (H 2 O), the free energy for the unfolding of the monomeric intermediate; ÁG(H 2 O), the total free energy of unfolding; and m 1 , m 2 and m, the coef®cients of dependence of these free energies on the concentration of urea (Park & Bedouelle, 1998). Here, we monitored the unfolding of the TyrRS(Á1) derivatives that carried the single mutations T51P, I52L or L105V, or the double mutation I52L-L105V, by their intensities of¯uorescence in identical exper- Table 2.…”