title: Conformation and reactivity changes induced by N-methylkirromycin (aurodox) in elongation factor Tu.
abstract: Kirromycin and related antibiotics inhibit protein synthesis in bacteria by acting on elongation factor Tu (EF-Tu). We have studied the effects of N-methylkirromycin (aurodox) on some molecular properties of this protein. The binding of the antibiotic causes a dramatic variation in the protein fluorescence emission spectrum with the appearance of a new maximum at around 340 nm. Addition of aurodox to trypsinized EF-Tu resulted in an emission spectrum similar to that of the denatured intact factor. Fluorescence lifetime analysis performed by a multifrequency phase fluorometer indicated that the fluorescence emission of the factor is heterogeneous with the major component having a lifetime near 4.8 ns in the absence and 6.6 ns in the presence of the antibiotic. These results were interpreted in terms of an antibiotic-induced environmental modification of the unique tryptophan residue of the protein leading to an increase in its quantum yield. However, aurodox did not modify the solvent exposure of this residue, as judged by fluorescence quenching experiments. Moreover, 1-anilino-8-naphthalenesulfonate (ANS) binding studies, as well as analysis of the protein reactivity toward the sulfhydryl group reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB), showed that, in the presence of aurodox, the behavior of the EF-Tu-GDP complex nears that of EF-Tu.GTP. These results strongly support the hypothesis that aurodox not only confers a "GTP-like" conformation to the EF-Tu.GDP complex but also produces a less stable folding of the protein around the tryptophan residue that may contribute to the multiple functional effects of this antibiotic.
authors.affiliation: Department of Biochemistry and Biophysics, University of Naples, Napoli, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Balestrieri
authors.forename: A
authors.initials: A
authors.lastname: Giovane
authors.forename: L
authors.initials: L
authors.lastname: Quagliuolo
authors.forename: L
authors.initials: L
authors.lastname: Servillo
authors.forename: G
authors.initials: G
authors.lastname: Chinali
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000899:Aurodox / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D000900:Anti-Bacterial Agents; D000899:Aurodox; D006153:Guanosine Diphosphate; D010444:Peptide Elongation Factor Tu