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 alpha-helices in tyrosyl-tRNA synthetase (TyrRS) from the thermophile Bacillus stearothermophilus. Its mechanism of evolution was characterized. Four residues of this cluster are not conserved in TyrRS from the mesophile Escherichia coli. The corresponding mutations were constructed in TyrRS(Delta1), a derivative of TyrRS from B. stearothermophilus in which the anticodon binding domain is deleted. Mutations I52L (i.e. Ile52 into Leu), M55L and L105V did not affect the activity of TyrRS(Delta1) in the pyrophosphate exchange reaction whereas T51P increased it. The kinetic stabilities of TyrRS(Delta1) and its mutant derivatives at 68.5 degreesC were determined from experiments of irreversible thermal precipitation. They were in the order L105V<I52L<T51P<Wild Type</=M55L; mutation I52L partially compensated L105V in these experiments whereas M55L was coupled neither to I52L nor to L105V. Mutations I52L and L105V affected the stability of the dimeric TyrRS(Delta1) at different steps of its unfolding by urea, monitored under equilibrium conditions by spectrofluorometry or size exclusion chromatography. I52L destabilized the association between the subunits even though residue Ile52 is more than 20 A away from the subunit interface. L105V destabilized the monomeric intermediate of unfolding. The two mutational pathways, going from the wild-type TyrRS(Delta1) to the I52L-L105V 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 pathway contained a destabilizing step followed by a stabilizing step. Mutation I52L allowed L105V along the first pathway and compensated it along the second pathway. Thus, the effects of I52L and L105V on stability depended on the structural context. The gain in activity due to T51P was at the expense of a slight destabilization.
Park
Y C
YC
Unité de Biochimie Cellulaire, Institut Pasteur, 28 rue du Docteur Roux, Paris Cedex 15, 75724, France.
Guez
V
V
Bedouelle
H
H
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D019281:Dimerization; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006358:Hot Temperature; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000187:drug effects; D014508:Urea / Q000494:pharmacology