Interaction studies between elongation factor Tu and anthraniloyl-fluorescent analogues of guanyl nucleotides

Eur J Biochem. 1995 Jan 15;227(1-2):428-32. doi: 10.1111/j.1432-1033.1995.tb20405.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence Polarization
  • Fluorescent Dyes / chemistry*
  • Guanosine Diphosphate / analogs & derivatives*
  • Guanosine Diphosphate / chemistry
  • Kinetics
  • Peptide Elongation Factor Tu / chemistry*
  • ortho-Aminobenzoates / chemistry*

Substances

  • 3'-O-anthraniloylguanosine diphosphate
  • Fluorescent Dyes
  • ortho-Aminobenzoates
  • Guanosine Diphosphate
  • Peptide Elongation Factor Tu