Examination of elongation factor Tu for aluminum fluoride binding sites using fluorescence and 19F‐NMR methodologies

Abstract

This article reports on a comparison or the interaction of AP³⁺ and F⁻ with two GTP‐binding proteins, elongation factor Tu (EF‐Tu) and the hormone sensitive regulatory protein (G protein) G_oα. The methodologies chosen to elucidate possible between protein and aluminum fluoride were fluorescence spectroscopy and nuclear magnetic resonance (¹⁹F‐NMR). Both proteins have tryptophan residues near their nucleotide binding sites, the purported site of aluminum fluoride interaction. It has been assumed for G proteins (including G_oα) that aluminum fluoride, in the presence of Mg²⁺, mimics the magnesium coordinated γ‐phosphate group for the GDP‐form of the protein and shifts the protein's conformation toward the active GTP‐form. Indeed, changes in intrinsic fluorescence of G_oα affected by aluminum fluoride are observed. The presence of aluminum fluoride did not affect the intrinsic fluorescence, spectra of lifetimes, of EF‐Tu·GDP. ¹⁹F‐NMR was then used to directly test for bound F⁻. Fluoride alone or in the presence of either protein gave a single ¹⁹F‐NMR peak at −10 ppm, characteristic of free F⁻. With the addition of aluminum to the protein and F⁻ samples a second peak, shifted upfield from the first to −29 ppm, was observed for G_oα·GDP. This second peak, which has been assigned to protein‐bound F⁻, was not observed for EF‐Tu·GDP. These observations show that the interaction of Al³⁺ and F⁻, in the presence of Mg²⁺, may be quite different between the hormone‐sensitive G proteins, which bind aluminum fluoride, and the GTP‐binding proteins as a whole, which include EF‐Tu. Care must therefore be exercised when structural data on the elongation factor, specifically on the nucleotide site, are used to interpret data or compose models intended to describe the hormone‐sensitive regulatory G proteins.