Table 5.

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 S100P 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	5.1 ± 0.3 (11 ± 1)	−1.1 ± 0.1	−1.7 ± 0.1	286 ± 43f	5.6 ± 0.9f
TRTKM1	2.0 ± 0.5 (7 ± 3)	−0.9 ± 0.1	−1.4 ± 0.1	135 ± 18	6.8 ± 1.9
TRTKM5	56.2 ± 4.0 (100 ± 23)	−1.2 ± 0.1	−1.5 ± 0.1	-	-
TRTKM6	7.4 ± 1.0 (16 ± 4)	−0.9 ± 0.1	−1.6 ± 0.1	-	-
TRTKM9	7.1 ± 0.5 (30 ± 10)	−1.0 ± 0.1	−1.7 ± 0.1	-	-
TRTKM10	43.1 ± 3.9 n.d.	−1.3 ± 0.1	−1.3 ± 0.1	32 ± 11g	0.07 ± 0.03g
TRTKM11	31.7 ± 0.5 n.d.	−1.8 ± 0.1	−1.5 ± 0.1	151 ± 25h	0.6 ± 0.1h

^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) using the average value from homology models based on the human S100P dimer binding to two peptides using PDB:3IQQ ⁽⁴⁴⁾.

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

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

^fk_off value is within error of the dead-time, but is consistent with the temperature-dependent slope of the rate constant.

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

^hk_off value was measured at 20°C. k_on value was calculated for the corresponding temperature.