Spectroscopic studies of Asparaginyl-tRNA synthetase from Entamoeba histolytica.

Aminoacyl-tRNA synthetases (aaRSs) play an important role in catalyzing the first step in protein synthesis. Asparaginyl-tRNA synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic pathogen that infects humans. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45˚C before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. Isothermal Calorimetry study revealed that ligand binding is mainly driven by hydrophobic interaction. Acrylamide quenching studies and lifetime measurements show the existence of two classes of tryptophan. This observation correlates with the unfolding data, wherein a red-shift in the emission maximum is seen due to better exposure of tryptophan residues to the solvent while unfolding.