Table 2.

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

S-peptide	Keq (M-1)	ΔΔG0 (kJ/mol)	kon (M-1 s-1)	ΔΔG0‡ (kJ/mol)	ϕ-value
WT	(3.6 ± 0.1)·106		(4.4 ± 0.2)·105
WT pH 8.5	(1.2 ± 0.1)·107	−3.0	(9.5 ± 0.6)·105
WT 1% DMSO	(2.2 ± 0.1)·106	1.2	(3.4 ± 0.1)·105
WT, low I*	(3.0 ± 0.1)·106	0.4	(3.7 ± 0.3)·105		-
A5F	(2.6 ± 0.1)·106	0.8	(4.2 ± 0.2)·105	0.12	n.w.d.
A6F	(3.6 ± 0.2)·105	5.7	(3.9 ± 0.2)·105	0.25	0.05
K7F	(1.9 ± 0.1)·106	1.7	(5.7 ± 0.4)·105	−0.66	< 0
F8Y†	(2.3 ± 0.3)·104	12.5	(3.4 ± 0.1)·105	0.61	0.05
F8W	(1.6 ± 0.2)·105	7.7	(5.0 ± 0.2)·105	−1.79	< 0
F8Nal	(1.1 ± 0.1)·106	3.5	(1.5 ± 0.1)·106	−2.42	< 0
E9Q	(5.1 ± 0.2)·106	−0.9	(5.7 ± 0.4)·105	−0.67	n.w.d.
E9F	(4.0 ± 0.2)·106	−0.3	(1.6 ± 0.1)·106	−3.16	< 0
R10F‡	(7.7 ± 0.3)·105	2.6	(5.8 ± 0.3)·105	−0.70	< 0
Q11F‡	(1.3 ± 0.1)·106	1.3	(3.4 ± 0.1)·105	0	0
H12A	(3.1 ± 0.1)·104	11.8	(2.7 ± 0.5)·106	−4.52	< 0
H12F	(1.4 ± 0.2)·105	8.0	(3.5 ± 0.9)·106	−5.15	< 0
Nle13A	(2.2 ± 0.8)·104	12.6	(2.3 ± 0.1)·105	1.59	0.14
Nle13F	(2.4 ± 0.1)·105	6.7	(5.7 ± 0.1)·105	−0.65	< 0
D14F	(1.1 ± 0.1)·105	8.6	38 ± 9	28.9	2.7

^*Low ionic strength (I) conditions were 10 mM NaOAc, pH 6.0, 25 °C resulting in I = 10 mM.

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

^‡Kinetics measured in the presence of 1% DMSO to increase solubility of the S-peptide variant (see Fig. S5).