Table 3.

Summary of the dissociation constants, the changes in the heat capacity upon binding, and the kinetic rate constants of the TRTK12 wild-type peptide and alanine variants binding to S100A1 at 25°C.

Peptide	Kda μM	ΔCp, expb kJ·mol−1·K−1	ΔCp, calcc kJ·mol−1·K−1	koff (s−1)d	kon (1×107 M−1·s−1)e
TRTK12	3.4 ± 1.0 (1 ± 0.5)	−1.6 ± 0.1	−1.0 ± 0.1 (−1.6 ± 0.1)	44 ± 8	1.2 ± 0.3
TRTKM1	1.0 ± 0.2 (0.5 ± 0.1)	−2.0 ± 0.1	−0.7 ± 0.1 (−1.4 ± 0.1)	6 ± 2f	0.6 ± 0.2f
TRTKM5	11.7 ± 1.0 (6 ± 2)	−1.7 ± 0.2	−0.7 ± 0.1 (−1.5 ± 0.1)	77 ± 22	0.7 ± 0.2
TRTKM6	5.3 ± 3.0 (7 ± 3)	−2.2 ± 0.1	−0.9 ± 0.1 (−1.7 ± 0.1)	87 ± 13	1.6 ± 1.0
TRTKM9	3.1 ± 1.0 (0.6 ± 0.1)	−1.5 ± 0.1	−1.4 ± 0.1 (−1.7 ± 0.1)	97 ± 21g	2.7 ± 0.8g
TRTKM10	9.9 ± 1.0 (27 ± 15)	−1.7 ± 0.1	−0.7 ± 0.1 (−1.3 ± 0.1)	63 ± 6g	1.7 ± 0.4g
TRTKM11	4.5 ± 1.9 (2 ± 1)	−1.5 ± 0.1	−1.3 ± 0.1 (−1.5 ± 0.1)	71 ± 12	1.6 ± 0.7

^aDissociation constants obtained using ITC. Values in parentheses were obtained using emission fluorescence assays.

^bThe experimentally determined changes in the heat capacity upon binding (Figure 3).

^cStructure-based calculations (see Eq. 7) homology models based on the human S100A1 dimer binding to two peptides using PDB: 2KBM ⁽³²⁾ or (3IQQ) ⁽⁴⁴⁾.

^dk_off values obtained using fluorescence stopped-flow spectroscopy (Eq 9).

^ek_on values were calculated using k_off rate constants and the dissociation constants obtained from ITC.

^fk_off value was measured at 5°C. k_on value was calculated for the corresponding temperature.

^gk_off value was measured at 15°C. k_on value was calculated for the corresponding temperature.