Tryptophan activation activity of a minimal TrpRS catalytic domain

Abstract

A ~130 residue fragment of B. stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) has been constructed by deleting the first connecting peptide, CP1, from the Rossmann fold and the C-terminal anticodon-binding domain. Moreover, a cycle of protein design has been used to “heal” scars created by removing the CP1 peptide. Characterizing the residual catalytic activity of this redesigned fragment is complicated by the fact that its activity is ~10−3 times that of the intact enzyme, making detection against contaminating chromosomal TrpRS problematic, even using FLAG-tagged fragments. We have addressed this problem in two ways. First, we have renatured the fragment from inclusion bodies, to purify it away from contaminating native activity. Second, we have mutated active-site residue D146 to alanine, to implicate the fragment directly. Both the redesigned fragment and the D146A mutant have catalytic activity above the essentially flat background measured for material renatured from inclusion bodies made with cells transformed with an empty vector. Moreover, the D146A mutant activity differs from that of the wild-type fragment. Significantly different specific activities for these two minimal catalytic domain fragments would provide strong evidence that analogous fragments could have had sufficient amino acid activating activity to support natural selection of a primitive translation apparatus. Supported by NIGMS 48519.