Expression and purification of threonyl tRNA synthetase RNA binding domain for heteronuclear NMR studies

Abstract

Heteronuclear NMR is a straightforward technique for the analysis of contact areas between one protein and its substrates. We have chosen this approach to study the interaction of threonyl-tRNA synthetase (ThrRS) with its two substrates. ThrRS forms a complex with the anticodon loop of ^ThrtRNA and also with a part of its mRNA, the second interaction being involved in the regulation of ThrRS expression. In these two cases, the interacting part of ThrRS is mostly limited to its C-terminal domain. A two-step study has been conducted: using a first genetic construction, we have validated our approach, which was then modified to improve the solubility and the stability of the recombinant domain. The latter construct was used to prepare an 15N labelled sample which gave heteronuclear NMR spectra of sufficient quality for structure and interaction studies.