Direct determination of the association constant between elongation factor Tu X GTP and aminoacyl-tRNA using fluorescence

Abstract

We have investigated the formation of the aa-tRNA X EF-Tu X GTP ternary complex spectroscopically by monitoring a fluorescence change that accompanies the association of EF-Tu X GTP with Phe-tRNAPhe-F8, a functionally active analogue of Phe-tRNAPhe with a fluorescein moiety covalently attached to the s4U-8 base. With this approach, the protein-nucleic acid interaction could be examined by direct means and at equilibrium. The fluorescence emission intensity of each Phe-tRNAPhe-F8 increased by 36-55% upon association with EF-Tu X GTP, depending on the solvent conditions. Thus, when Phe-tRNAPhe-F8 was titrated with EF-Tu X GTP, the extent of ternary complex formation was determined from the increase in emission intensity. A nonlinear least-squares analysis of the titration data yielded a dissociation constant of 0.85 nM for the ternary complex in 50 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (pH 7.6), 10 mM MgCl2, and 50 mM NH4Cl, at 6 degrees C. The delta H degrees of this interaction, determined by the temperature dependence of Kd, was -16 kcal/mol; the delta S degrees was therefore -16 cal mol-1 deg-1 at 6 degrees C in this buffer. In a more physiological polycation-containing solvent ("polymix"), the Kd was 4.7 nM. The ionic strength dependence of ternary complex formation showed that a minimum of two salt bridges and a substantial nonelectrostatic contribution are involved in the binding of aa-tRNA to EF-Tu. The affinities of unmodified aa-tRNAs for EF-Tu X GTP were determined by their abilities to compete with the fluorescent aa-tRNA for binding to the protein.(ABSTRACT TRUNCATED AT 250 WORDS)