Intrinsic fluorescence of elongation factor Tu in its complexes with GDP and elongation factor Ts.

David M Jameson,John F Eccleston,Enrico Gratton

doi:10.1021/bi00387a023

Abstract

The intrinsic fluorescence properties of elongation factor Tu (EF-Tu) in its complexes with GDP and elongation factor Ts (EF-Ts) have been investigated. The emission spectra for both complexes are dominated by the tyrosine contribution upon excitation at 280 nm whereas excitation at 300 nm leads to exclusive emission from the single tryptophan residue (Trp-184) of EF-Tu. The fluorescence lifetime of this tryptophan residue in both complexes was investigated by using a multifrequency phase fluorometer which achieves a broad range of modulation frequencies utilizing the harmonic content of a mode-locked laser. These results indicated a heterogeneous emission with major components near 4.8 ns for both complexes. Quenching experiments on both complexes indicated limited accessibility of the tryptophan residue to acrylamide and virtually no accessibility to iodide ion. The quenching patterns exhibited by EF-Tu-GDP and EF-Tu X EF-Ts were, however, different; both quenchers were more efficient at quenching the emission from the EF-Tu x EF-Ts complex. Steady-state and dynamic polarization measurements revealed limited local mobility for the tryptophan in the EF-Tu x GDP complex whereas formation of the EF-Tu x EF-Ts complex led to a dramatic increase in this local mobility.

Highlights

The emission spectra for both complexes are dominated by the tyrosine contribution upon excitation at 280 nm whereas excitation a t 300 nm leads to exclusive emission from the single tryptophan residue (Trp-184) of elongation factor T u (EF-Tu)
Since this preparation is heterogeneous with respect to the bound nucleotide, it was converted to EF-TusGDP as described by Eccleston (1984)
The slight blue shift and decreased F W H M of the EF-Tu-elongation factor Ts (EF-Ts) complex compared to the EFTwGDP complex suggest an increased tyrosine contribution in the emission relative to the tryptophan contribution due to the additional tyrosine residues in EF-Ts

Summary

Introduction

Previous spectroscopic investigations of EF-Tu, using methodologies such as ANS binding, ORD, CD, and total intrinsic fluorescence signals [reviewed by Kaziro (1978)], have been directed toward detecting differences between the EFTu-GDP and EF-TueGTP complexes These studies have not addressed questions pertaining to specific structural changes in the protein complexes. Steady-state (emission spectra, polarization, and quenching) and dynamic (time-resolved measurements) fluorescence studies of the single tryptophan residue afford us information about the local environment of this fluorophore, its motional properties and accessibility to solvent, and general hydrodynamic properties of the protein complexes. This information may be important in assessing the details of conformational changes and dynamic aspects in the EF-TusGDP and EFTwEF-TScomplexes. The presence of 10 tyrosine residues in EF-Tu and 3 in EF-Ts renders the tyrosine emission properties, per se, too complex for most quantitative interpretations, the possibility of tyrosine to tryptophan energy transfer can provide an additional probe of protein conformation in the complexes

Methods

Results

Conclusion