Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Investigations of Translational GTPases using Isothermal Titration Calorimetry Reveal G-nulceotide Dependent Structural Rearrangements
GTPases oscillate between their GTP- and GDP-bound states via regulated cycles of GTP hydrolysis and exchange of GDP for GTP. The interaction between the GTPase and the G nucleotide is mediated by switch 1 and 2 (sw 1 and sw 2) regions of the GTPase domain. The nature of the bound nucleotide is believed to be the main factor regulating the functional state of the protein, regulating its on / off modes: off in the apo and GDP-bound state, and on in the GTP-bound state.
We used isothermal titration calorimetry to investigate the GTPase cycle of a number of GTPases involved in translation (trGTPases): IF2, EF-G, and eRF3. For these we determined Kd and thus Gibbs energy (ΔG0), enthalpy (ΔH0), entropy (ΔS0) and change in heat capacity (ΔCp= d(ΔH)/dT) of the interaction between the protein and the G nucleotides. The last parameter (ΔCp) is directly proportional to change in the solvent accessible area of the protein and thus reflects the extent of the structural rearrangement, bridging the gap between the physical chemistry and structural biology descriptions of the system.
Our ΔCp data suggest that in the case of EF-G, GTP binding promotes ordering of the sw 1 and sw 2 regions, but GDP does not (1) (Fig. 1) as indicated by the 250 cal·mol-1·K-1 ΔCp difference in GDP and GTP binding. In the IF2 case, in addition to the GTP-mediated ordering of the sw 1 and sw 2 regions that cause the 290 cal·mol-1·K-1 difference between GDP and GTP binding, both GTP and GDP promote a large-scale rearrangement in the protein, suggested by the large ΔCp that binding of either nucleotide causes (Fig. 1). Finally, in the eRF3 case, binding of eRF1 is necessary for GTP binding (2), which promotes a large-scale rearrangement of the eRF1:eRF3 complex (Fig. 1).
The results presented here demonstrate that despite the common functional cycle, different trGTPases do have significant differences in the way they are regulated by G nucleotides.
Figure 1: Enthalpy of binding of GDP (empty circles) and GTP (filled circles) to trGTPases as a function of the temperature (°C) at pH 7.5.
References and Footnotes
Vladimir A. Mitkevich1
1Engelhardt Institute of Molecular Biology