Table 1.

Transition temperatures for EI(H189A) and wild-type, dephospho-EI in the absence and presence of effectors as measured by DSC or intrinsic Trp fluorescence changes (FI) as a function of increasing temperaturea

		F1	DSC
Protein (mg/mL)	Effector(s) present (mM)	Tm (°C)	Tm1 (°C)	Tm2 (°C)
EI(H189A), 02.0	—	46.8	45.4	54.4
EI(H189A), 0.15	Hepes	48.0	48.9	54.3
EI(H189A), 0.29	5 Pyr	48.0	45.6	53.4
EI(H189A), 0.21	5 Pyr/2 Mg2+	50.5	nt	50;53
EI(H189A), 0.19	5 Pyr/0.2 PEP/2 Mg2+	59.9	54.8	60;61
EI(H189A), 0.26	5 Pyr/0.5 PEP/2 Mg2+	63.9	52.5	62;63
EI(H189A), 0.21	5 Pyr/1 PEP/2 Mg2+	63.3	53.7	59;63
EI(H189A), 0.29	0.2 PEP/2 Mg2+	63.3	53.5	60;63
EI(H189A), 0.22b	1 PEP/2 Mg2+	64.6	nt	62;63
EI(H189A), 0.29	0.2 PEP/10 Mg2+	64.4	—	—
EI(H189A), 0.29	0.2 PEP/10 Mg2+/Hepes	—	52.9	62;65
EI(wt), 0.15	—	43.0	46.4	55.4
E(wt), 0.23	Hepes	—	48.8	54.7
EI(wt), 0.20	0.65 PPyr/2 Mg2+	63.2	nt	61;63

^a Proteins are in Buffer A containing 20 mM K-phosphate (pH 7.5) or when Hepes is indicated in Buffer B containing 20 mM Hepes (pH 7.5). The rate of temperature increase is 30°C/h. Transition temperatures are from two-state analysis of Trp fluorescence changes (F1) as a function of increasing temperature for C-terminal domain unfolding and from two independent two-state analyses of DSC data in the absence of effectors when two endotherms are resolved. When endotherms in DSC overlap (under T_m²), deconvolutions have assumed a dependent model of two, two-state transitions for analysis of DSC data and the two T_m values are given. When pyruvate is present in mixtures of effectors, endotherms 1 and 2 represent different populations of N- and C-terminal domain unfolding, and deconvolutions of DSC data give one transition for decoupled N-terminal domain under T_m¹ and two transitions under T_m² for coupled N- and C-terminal domain unfolding. The abbreviation ‘nt’ means that no transition for a resolved lower temperature endotherm with T_m¹ is observed.