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 .
1. Answer the following questions based on the ITC thermograms for binding of GTP to SelB (A) and GDP to SelB (B) below.
a. The titration curves above appear sigmoidal in contrast to the hyperbolic saturation binding curve you drew for a simple M + L binding equilibrium. Sigmoidal ML vs L binding plots are often associated with cooperative binding interactions. Why are these plots NOT hyperbolic even they the enthalpy changes can be best modeled by a simple, non-cooperative binding of M and L with a stoichiometry of 1?
b. Is the binding exothermic or endothermic? What is the sign of DH?
b. Which one proceeds with a lower dissociation constant (Kd)?
c. Assuming that the binding is favored, which one has a more negative DG0? Assume equal concentrations of SelB in the sample cell. Which one is more favored under standard condition?
d. The change in Gibbs Free energy is also related to the change in enthalpy and entropy as shown in the equation below:
Can you deduce from simple inspection of the curves above if DS0 is <, =, or >? Explain.
The ITC thermograms comparing the binding of GTP (A) and two analogs of GTP, GTPγS (B), GDPNP (C), are shown below
d. Again assuming similar concentration of SelB in the sample C, compare the Kds for the interaction of GTPγS and GDPN to SelB.
e. The structures of GTP and the two analogs are shown below. Suggests a reason that the investigators used the analogs in their studies.
f. The ITC thermograms be fit to a theoretical model for M and L binding with a stoichiometry of one. From this data fit, Kd can be determined. How would you calculate the DS0 for the interaction of SelB and GXP?
The thermodynamic parameters of SelB binding to guanine nucleotides at different temperatures are shown below.
g. Do they confirm your answers to b and d above?
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