Guanidine Hydrochloride Mediated Denaturation of E. coli Alanyl-tRNA Synthetase: Identification of an Inactive Dimeric Intermediate

Abstract

E. coli Alanyl-tRNA synthetase (AlaRS) not only catalyzes tRNA charging but also can bind to its own promoter DNA sequence and repress its own transcription. It exists as a dimer in its native form and so far this is the only aminoacyl-tRNA synthetase whose full length structure is unresolved. Guanidine hydrochloride mediated unfolding of AlaRS has been studied under equilibrium conditions using various spectroscopic techniques such as intrinsic tryptophan fluorescence, 1-anilino-8-naphthalene-sulfonic acid binding, near and far-UV circular dichroism and analytical ultracentrifugation. These studies revealed that in presence of gdnHCl AlaRS unfolded in a multistep pathway. At 0.8 M gdnHCl, AlaRS formed a molten globule like intermediate, which was enzymatically inactive. Further characterization of this intermediate proved that there was no oligomer breakdown at this denaturant concentration. This study clearly indicates that unlike many other oligomeric proteins AlaRS unfolding does not follow the hierarchical model as in this enzyme tertiary structure gets disrupted well before the disruption of quaternary interaction.

Urea Unfolding Study ofE. coliAlanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

“…We examined the fluorescence emission spectrum of the wild type and three mutant alaRSs at different urea concentrations. At 0 M urea WT-alaRS showed an emission maximum at 336 nm, which is consistent with our previous study [ 29 ]. With increasing urea concentration the emission maximum was red-shifted and finally levelled off around 350 nm at 8 M urea.…”

“…To understand this in more detail, we had determined the sedimentation coefficients at different urea concentartions. E. coli alaRS exists as a dimer in the absence of any denatutants with a sedimentation coefficient of ~6.1 S, consistent with the previous report as dimer [ 9 , 29 ]. In the presence of 8 M urea, sedimentation value of ~3 S was obtained, which corresposponds to the presesnce of only monomeric species.…”

Adaptation of a eukaryote-like ProRS to a prokaryote-like tRNAPro

“…Dithiothreitol (DTT) is a powerful reducing agent that induces acute ER stress by disrupting the redox conditions required to form disulfide bridges in proteins ( 38 ). Urea and guanidine hydrochloride (GnHCl) can induce denaturation of proteins ( 39 , 40 ). To assess whether BtProRS1 and BtProRS2 exhibit different sensitivities toward these stresses and denaturants, we pretreated the enzymes with these agents for 10 min before analyzing their aminoacylation activity.…”