Table 2.

Thermodynamic parameters of eIF5B binding to GDP and GTP at different temperatures and in presence or absence of Mg²⁺ ions

Construct	Ligand	MgCl2 (mM)	T (°C)	Kd (µM)	ΔH (cal/mol)	ΔG (kcal/mol)	TΔS (kcal/mol)
Ct-eIF5B(517C)	GDP	2.5	5	2.09	−6286	−7.23	1.0
	GDP	2.5	10	1.92	−5057	−7.4	2.3
	GDP	2.5	15	2.61	−5240	−7.36	2.0
	GDP	2.5	20	2.9	−5520	−7.43	1.9
	GDP	2.5	25	3.3	−6058	−7.48	1.4
	GDP	2.5	30	3.45	−6650	−7.58	0.9
	GDP	0	5	0.8	−1855	−7.76	5.9
	GDP	0	10	1.0	−2385	−7.77	5.4
	GDP	0	15	1.14	−2805	−7.84	5.0
	GDP	0	25	1.96	−3912	−7.79	3.9
	GTP	2.5	5	4.21	−5346	−6.84	1.5
	GTP	2.5	10	4.83	−6192	−6.89	0.7
	GTP	2.5	15	5.68	−7033	−6.92	0.12
	GTP	2.5	20	6.02	−7423	−7.0	−0.6
	GTP	2.5	25	6.20	−8426	−7.1	−1.3
	GTP	2.5	30	7.04	−9344	−7.03	−2.2
Ct-eIF5B(517–858)	GTP	2.5	5	1.34	−4860	−7.47	2.6
	GTP	2.5	10	1.58	−7822	−7.52	−0.3
	GTP	2.5	20	2.12	−12,985	−7.61	−5.4
	GTP	2.5	30	4.07	−18,810	−7.48	−11.3

All measurements were performed two to four times; for GTP binding to both constructs and for GDP binding in the presence of Mg²⁺ the experiments were done with two independent purifications of the respective construct; for GDP binding in the absence of Mg²⁺, the experiments were done with protein from one purification.

K_d, dissociation equilibrium constant; calculated as 1/K_a.

K_a, association equilibrium constant; standard deviation did not exceed ± 15%.

ΔH, standard enthalpy change; standard deviation did not exceed ± 10%.

ΔG, Gibbs energy; calculated from equation ΔG = −RTlnK_a.

TΔS, standard entropy change; calculated from equation ΔG = ΔH − TΔS.