Background:
Aminoacyl-tRNA synthetases play an important role in catalyzing the first
step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then
transported to the growing polypeptide chain. 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 eukaryotic
pathogen that infects the large intestines of humans. It is a major cause of dysentery and has
the potential to cause life-threatening abscesses in the liver and other organs making it the second
leading cause of parasitic death after malaria. 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. It is
a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short
N-terminal as compared to its human and yeast counterparts.
Objective:
Our study focusses to understand certain structural characteristics of Ehis-AsnRS using
biophysical tools to decipher the thermodynamics of unfolding and its binding properties.
Methods:
Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification
was performed using Ni-NTA affinity chromatography, following which the protein was used for
biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism,
differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies
were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for
far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4
µM protein was used for the rest of the experiments.
Results:
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 45ºC 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.
Conclusion:
This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two –
state process and does not involve any intermediate; unfolding occurs directly from native dimer to
unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective
quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide
range of pH.
Steady-state fluorescence and circular dichroism of trout hemoglobins I and IV interacting with tributyltin
