Table 1.

Effect of single thioxo-bonds on stability and association kinetics of the S-peptide/S-protein complex at pH 6.0, 50 mM NaOAc, 100 mM NaCl, 25 °C

Position of thioxo-bond	Keq (M-1)	ΔΔG0* (kJ/mol)	kon (·105 M-1 s-1)	ΔΔG0‡* (kJ/mol)	ϕ-value†
WT	(3.6 ± 0.1)·106	-	(4.4 ± 0.2)·105		-
Lys1	(1.3 ± 0.1)·106	2.5	n.d.
Glu2	(1.1 ± 0.1)·106	2.9	n.d.
Thr3	(6.1 ± 0.1)·104	10.1	(3.0 ± 0.1)·105	0.90	0.09
Ala4‡	(1.4 ± 0.1)·106	2.3	(4.4 ± 0.1)·105	0	-
Lys7	(1.3 ± 0.1)·104	13.9	(4.1 ± 0.3)·105	0.17	0.01
Phe8§	(6.8 ± 0.9)·103	15.5	(2.2 ± 0.1)·105	1.7	0.10
Glu9	(4.0 ± 0.5)·104	11.1	(5.1 ± 0.4)·105	−0.41	−0.04
Arg10	(6.5 ± 0.1)·104	9.9	(4.1 ± 0.3)·105	0.14	0.01
His12§,¶	(1.3 ± 0.3)·103	19.6	n.d.	-	n.d.
Ser15	(2.9 ± 0.1)·105	6.2	(3.6 ± 0.4)·105	0.46	0.07

^*ΔΔG⁰ represents the difference in free energy between a variant and the WT protein, which has a ΔG⁰ of -37.4 kJ/mol. ΔG⁰ was calculated from the equilibrium constant using ΔG⁰ = -RT ln K_eq.

^†Errors in ϕ-values depend on the errors in K_eq and k_on as well as on ΔΔG⁰ and are ≤ 0.05 for the variants used for ϕ-value analysis.

^‡ΔΔG⁰ too small to determine an accurate ϕ-value (8).

^§Stability of the complex was determined by an activity assay (see SI Text and Fig. S3).

^¶Thioxo His12 is strongly destabilized, which does not allow measurement of the association kinetics by stopped-flow fluorescence.