The anticodon-binding domain of tyrosyl-tRNA synthetase: state of folding and origin of the crystallographic disorder.
The C-terminal domain (residues 320-419) of tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus is disordered in the crystal structure. Its function consists of binding the anticodon of tRNA(Tyr). We undertook to characterize its conformational state. A hybrid between the C-terminal fragment and a His-tag sequence was constructed and purified in large amounts. Analyses by mass spectrometry and analytical ultracentrifugation showed that the C-terminal fragment, thus purified, was not degraded and that it neither dimerized nor aggregated. Its far- and near-UV circular dichroism spectra revealed a high content in secondary structures and an asymmetrical environment of its aromatic residues. Each spectrum could be reconstructed by the difference between the corresponding spectra for the full-length TyrRS and for its N-terminal fragment. The Stokes radius of the C-terminal fragment, measured by size exclusion chromatography, indicated a condensed globular state. The fluorescence of ANS (a small hydrophobic probe) showed that the surface of the C-terminal fragment was more hydrophilic than that of a molten globule. These results on the C-terminal fragment and our previous observations that it can undergo cooperative transitions, demonstrated the following points: it is not in a disordered or molten globular state, it has a defined and stable three-dimensional structure, its structures are similar in its isolated and integrated forms, and the apparent disorder in the crystals of the full-length synthetase must be due to the flexibility of the polypeptide segment that links the N- and C-terminal domains. Thus, TyrRS has not evolved strong noncovalent interactions between its catalytic and anticodon-binding domains, contrary to the other synthetases.
Guez
V
V
Unité de Biochimie Cellulaire, CNRS-URA1129, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Nair
S
S
Chaffotte
A
A
Bedouelle
H
H
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000926:Anticodon / Q000737:chemistry* / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D000926:Anticodon; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D006639:Histidine; C027132:1-anilino-8-naphthalenesulfonate; D014448:Tyrosine-tRNA Ligase