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 0.8M 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.
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