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Interaction of a Fluorescent Analogue of GDP with Elongation Factor Tu: Steady-State and Time-Resolved Fluorescence Studies

  • Author(s): Eccleston, JF
  • Gratton, E
  • Jameson, DM
  • et al.
Abstract

A fluorescent derivative of GDP was prepared by the reaction of 2‘-amino-2’-deoxy-GDP with fluorescamine. This derivative binds tightly (KD ~ 4.5 X 10–8 M) to elongation factor Tu (EF-Tu) from Escherichia coli. The emission properties, including spectra, polarizations, and lifetimes, for fluoresca-mine-GDP free in solution and bound to EF-Tu are presented. Emission data on the fluorescamine-ethylamine conjugate are also given. A multifrequency phase and modulation lifetime study (using nine modulation frequencies over the range of 2–80 MHz) indicated that the emissions of these three systems were well characterized by single exponential decays corresponding to 1.45 ns for the fluorescamine-ethylamine derivative in buffer and to 7.74 and 11.03 ns for the fluorescamine-GDP derivative free in buffer and bound to EF-Tu, respectively. Multifrequency differential polarized phase fluorometry results indicated a rotational relaxation time of the protein-probe complex of approximately 88 ns; these data also indicated the lack of significant local motion for the probe. Addition of excess GDP to the EF-Tu-probe complex led to displacement of the fluorescamine-GDP derivative as evidenced by the change in both the steady-state and dynamic polarization values. The observed increase in fluorescence intensity upon displacement allowed us to follow the kinetics of the dissociation reaction; a dissociation rate constant of 5.0 X 10-3 s-1was determined. These results demonstrate the utility of this 2‘-amino-2’-deoxy-GDP analogue as a probe of guanosine nucleotide dependent systems. © 1987, American Chemical Society. All rights reserved.

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