title: Re-designed N-terminus enhances expression, solubility and crystallizability of mitochondrial protein.
abstract: Mitochondrial aminoacyl-tRNA synthetases are key enzymes in translation. They are encoded by the nuclear genome, synthesized as precursors in the cytosol and imported. Most are matured by cleavage of their N-terminal targeting sequence. The poor expression of mature proteins in prokaryotic systems, along with their low solubility and stability after purification are major obstacles for biophysical and crystallographic studies. The purpose of the present work was to analyze the influence of additives on a slightly soluble aspartyl-tRNA synthetase and of the N-terminal sequence of the protein on its expression and solubility. On the one hand, the solubility of the enzyme was augmented to some extent in the presence of a chemical analog of the intermediary product aspartyl-adenylate, 5'-O-[N-(L aspartyl) sulfamoyl] adenosine. On the other hand, expression was enhanced by extending the N-terminus by seven natural amino acids from the predicted targeting sequence. The re-designed enzyme was active, monodisperse, more soluble and yielded crystals that are suitable for structure determination. This result underlines the importance of the N-terminal residue sequence for solubility. It suggests that additional criteria should be taken into account for the prediction of cleavage sites in mitochondrial targeting sequences.
authors.affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 15 rue René Descartes, 67084 Strasbourg cedex, France. M.Sissler@ibmc-cnrs.unistra.fr
authors.forename: Agnès
authors.initials: A
authors.lastname: Gaudry
authors.forename: Bernard
authors.initials: B
authors.lastname: Lorber
authors.forename: Anne
authors.initials: A
authors.lastname: Neuenfeldt
authors.forename: Claude
authors.initials: C
authors.lastname: Sauter
authors.forename: Catherine
authors.initials: C
authors.lastname: Florentz
authors.forename: Marie
authors.initials: M
authors.lastname: Sissler
mesh_terms: D000595:Amino Acid Sequence; D001226:Aspartate-tRNA Ligase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D001226:Aspartate-tRNA Ligase