Examination of elongation factor Tu for aluminum fluoride binding sites using fluorescence and 19F-NMR methodologies.
This article reports on a comparison of the interaction of Al3+ and F- with two GTP-binding proteins, elongation factor Tu (EF-Tu) and the hormone sensitive regulatory protein (G protein) G0 alpha. The methodologies chosen to elucidate possible interactions 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 G0 alpha) that aluminum fluoride, in the presence of Mg2+ mimics the magnesium coordinated gamma-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 G0 alpha effected by aluminum fluoride are observed. The presence of aluminum fluoride did not affect the intrinsic fluorescence, spectra or 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 G0 alpha.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.
Hazlett
T L
TL
Department of Biochemistry and Biophysics, University of Hawaii, Honolulu 96822.
Higashijima
T
T
Jameson
D M
DM
D000535:Aluminum / Q000378:metabolism*; D017607:Aluminum Compounds; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli; D005459:Fluorides / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D017607:Aluminum Compounds; D001426:Bacterial Proteins; D000535:Aluminum; D019204:GTP-Binding Proteins; D010444:Peptide Elongation Factor Tu; D005459:Fluorides; C032311:aluminum fluoride