Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: Quantitative effects on activity, stability and dimerization

A dense cluster of eight residues was identified at the crossing of two alp ha-helices in tyrosyl-tRNA synthetase (TyrRS) from the thermophile Bacillus stearothermophilus. Its mechanism of evolution was characterized. Four res idues of this cluster are not conserved in TyrRS from the mesophile Escheri chia coli. The corresponding mutations were constructed in TyrRS(Delta 1), a derivative of TyrRS from B. stearothermophilus in which the anticodon bin ding domain is deleted. Mutations I52L (i.e. Ile52 into Leu), M55L and L105 V did not affect the activity of TyrRS(Delta 1) in the pyrophosphate exchan ge reaction whereas T51P increased it. The kinetic stabilities of TyrRS(Del ta 1) and its mutant derivatives at 68.5 degrees C were determined from exp eriments of irreversible thermal precipitation. They were in the order L105 V < I52L < T51P < Wild Type less than or equal to M55L; mutation I52L parti ally compensated L105V in these experiments whereas M55L was coupled neithe r to I52L nor to L105V. Mutations I52L and L105V affected the stability of the dimeric TyrRS(Delta 1) at different steps of its unfolding by urea, mon itored under equilibrium conditions by spectrofluorometry or size exclusion chromatography. I52L destabilized the association between the subunits eve n though residue Ile52 is more than 20 Angstrom away from the subunit inter face. L105V destabilized the monomeric intermediate of unfolding. The two m utational pathways, going from the wildtype TyrRS(Delta 1) to the I52L-L105 V double mutant through each of the single mutants were not equivalent for the stability of the monomeric intermediate and for the total stability of the dimer. One pathway contained two neutral steps whereas the other pathwa y contained a destabilizing step followed by a stabilizing step. Mutation I 52L allowed L105V along the first pathway and compensated it along the seco nd pathway. Thus, the effects of I52L and L105V on stability depended on th e structural context. The gain in activity due to T51P was at the expense o f a slight destabilization. (C) 1999 Academic Press.