Interaction studies between elongation factor Tu and anthraniloyl-fluorescent analogues of guanyl nucleotides.
A fluorescent analogue of GDP, the 3'-O-anthraniloyl-GDP (anl-GDP) was demonstrated to bind to the elongation factor Tu (EF-Tu) with an affinity even higher than that of the parent nucleotide. As a consequence of the binding, an increase in fluorescence anisotropy and an emission band arising from non-radiative energy transfer among the protein intrinsic fluorophores and the labelled nucleotide were observed. Therefore, it was possible to study the exchange kinetics and the equilibrium between the protein-bound labelled GDP and the natural nucleotide through modifications, occurring during the course of the reaction, of fluorescence anisotropy and non-radiative energy transfer. In this way, it was also easily proven that, in the presence of aurodox (N-methylkirromycin), an antibiotic impairing EF-Tu biological function, the exchange kinetics between the protein-bound labeled GDP and the natural nucleotide was faster. Moreover, it was also found that the labelled nucleotide is recognized as a substrate by pyruvate kinase, being converted by this enzyme, in the presence of phosphoenolpyruvate, into anl-GTP. Pyruvate kinase is also able to convert, in the presence of phosphoenolpyruvate, the complex EF-Tu.anl-GDP into the complex EF-Tu.anl-GTP. The fluorescence properties of the 3'-O-anthraniloyl-labeled guanyl nucleotides and their feature as excellent acceptors of fluorescence arising from protein intrinsic fluorophores, may make these compounds useful for structural and binding studies on guanosine-nucleotide-binding proteins.
Department of Biochemistry and Biophysics, II University of Naples, Italy.
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Giovane
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Balestrieri
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Balestrieri
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Servillo
D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D010444:Peptide Elongation Factor Tu / Q000737:chemistry*; D062367:ortho-Aminobenzoates / Q000737:chemistry*
C091920:3'-O-anthraniloylguanosine diphosphate; D005456:Fluorescent Dyes; D062367:ortho-Aminobenzoates; D006153:Guanosine Diphosphate; D010444:Peptide Elongation Factor Tu