Thermodynamics of the GTP-GDP-operated Conformational Switch of Selenocysteine-specific Translation Factor SelB. Alena Paleskava, Andrey L. Konevega and Marina V. Rodnina . The Journal of Biological Chemistry, 287, 27906-27912 (2012). doi: 10.1074/jbc.M112.366120 .
3. The main source of DCp changes on binding arise from loss of water of interacting water from the surfaces of protein and ligand on binding, with the main contribution arising from the hydrophoboic effect. Another less sizeable contribution to DCp would arise from burying polar residues with the ensuing changes in bound and bulk water interaction potentials. The following equation has been used to show the contribution to ΔCp from dissolution of solids compounds in water:
ΔCp = DcapDASAap + DcpDASAp,
where DASAap and DASAp are the apolar and polar solvent accessible surface areas changes, respectively. Model compounds give values of Dcap = 0.45 and Dcp= -0.26 per mol of SA (angstroms squared).
Table 2 below shows heat capacity changes and accessible surface area for SelB binding to guanine nucleotides. ΔCp, heat capacity change; obtained as ΔH/dT; ΔASAap and ΔASAp, changes in apolar and polar solvent-accessible surface areas assuming that all the changes were conferred by either apolar (AAap) or polar (AAp) residues, respectively with no contribution from the other. (Ex: for GTP in Hepes, 621/0.45=1380) The values for the buried area were converted into the numbers of amino acids that were removed from the surface using the average ASA for apolar (34 Å2) and polar (56 Å2) amino acids.
Figure 3 shows the heat capacity changes upon SelB interaction with guanine nucleotides. A, temperature dependence of binding enthalpy changes upon SelB interactions with GTP (●), GTPγS (○), GDPNP (■), and GDP (□). Standard deviations of measurements are given by error bars, which are smaller than the symbol size. B, bar representation of heat capacity changes upon SelB interactions with guanine nucleotides.
From graphs A and B and Table 2 above, compare the similarities and differences between the free and bound states of SelB with the different ligands.
Answer: GTP binding to SelB caused a large change in heat capacity, suggesting a major structural rearrangement corresponding to 41–43 amino acids that altered their contacts upon binding. With GTPγS, the conformational changes were somewhat smaller, indicating that 31–32 amino acids were affected by complex formation. The smallest changes were observed with GDP and GDPNP with 15–19 amino acids that changed their interaction partners upon interaction with SelB. The ITC measurements indicate that the structures of the GTP- and GDP-bound forms of SelB are different and that about 25 amino acids rearrange during the GTP-GDP conformational switch. Although it is usually assumed that all nonhydrolyzable GTP analogs can readily substitute for GTP, our findings suggest that a careful analysis of different GTP analogs is required to find the one that resembles the natural substrate most closely. The affinity of SelB for GDPNP is only two times lower than for GTP, but the values of both enthalpy and entropy changes resembled more those of GDP binding. Furthermore, SelB·GDPNP has a 10-fold lower affinity for Sec-tRNASec when compared with SelB·GTP, supporting the notion that GDPNP is not an authentic GTP analog for SelB. On the other hand, binding of GTPγS to SelB is surprisingly tight with aKd value 3–4 times smaller than that of SelB·GTP. Because other thermodynamic parameters are comparable for GTPγS and GTP complexes of SelB, we conclude that GTPγS is a better GTP analog for SelB than GDPNP. The general implication of these findings is that nonhydrolyzable GTP analogs are not in all cases faithful replacements for GTP, and thus, results obtained with the help of these analogs, including crystal structures, have to be interpreted with caution.
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