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

Abstract

E. coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in the dimerization process. The truncated N-terminal containing the first 700 amino acids (1–700) forms a monomeric variant possessing similar aminoacylation activity like wild type. A point mutation in the C-terminal domain (G674D) also produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild type enzyme. Urea induced denaturation of these monomeric mutants along with another alaRS variant (N461 alaRS) was studied together with the full-length enzyme 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. Aminoacylation activity assay after refolding from denatured state revealed that the monomeric mutants studied here were unable to regain their activity, whereas the dimeric full-length alaRS gets back similar activity as the native enzyme. This study indicates that dimerization is one of the key regulatory factors that is important in the proper folding and stability of E. coli alaRS.

Biophysical and Structural Characterization of Ribulose-5-phosphate Epimerase from Leishmania donovani

“…It is in corroboration with the earlier studies on the alanyl-tRNA synthetase from E. coli and the aspartyl-tRNA synthetase from L. donovani .…”

“… 30 Further, the secondary structure of Ld RPE revealed a moderate stability with no significant changes at lower concentrations of urea but complete loss in ellipticity at its higher levels. It is in corroboration with the earlier studies on the alanyl-tRNA synthetase from E. coli ( 31 ) and the aspartyl-tRNA synthetase from L. donovani . 32 Tryptophan fluorescence is widely used to probe the structural changes of protein during unfolding, 33 and it indicates that tryptophan residues of Ld RPE are located in a relatively hydrophobic milieu with only a small fraction on the protein surface.…”

Characterization of Cyclophilin from Thaumarchaeota Nitrosopumilus maritimus: Implications on the Diversity of Chaperone-like Activity in the Archaeal Domain

“…The unfolding experiment data obtained from CD were further fitted into a two-state equation ( Figure 7 C). 20 , 40 − 42 The obtained m (cooperativity) and Cm (mid-point of unfolding) from fitted data were used for the calculation of Δ G NU H 2 O , and the obtained value was 2.7 ± 0.97 kcal mol –1 ( Table S3 ).…”

“…The data were further fitted into a two-state equation to estimate the thermodynamic parameters of NmCyp ( Figure 10 and Table S3 ). 20 , 40 − 42 The mid-point of unfolding (Cm NU ) and cooperativity ( m ) for NmCyp is 0.75 ± 0.02 M and 4.45 ± 0.48 kcal mol –1 M –1 , respectively.…”

Characterization of aminoacyl-tRNA synthetase stability and substrate interaction by differential scanning fluorimetry

“…The aminoacylation reaction involves binding of the three substrates amino acid, tRNA, and ATP to each AARS to produce the final aminoacylated tRNA product. Traditionally, biophysical techniques to measure AARS enzyme stability and unfolding have included spectroscopic methods such as intrinsic tryptophan fluorescence in the presence of a denaturant like urea or guanidine-HCl, monitoring fluorescence with 1-anilino-8-naphthalene-sulfonic (ANS) dye, and circular dichroism [1–4]. An example of a classic method for determining a protein melting point without the use of exterior dyes is differential scanning calorimetry (DSC) [5,6].…”