. 2013 Jan 16;280(4):1018–1027. doi: 10.1111/febs.12094

Table 2.

Characterization of two-domain mutant proteins

1	2	3	4	5	6	7	8	9	10	11	12	13	14
	Kinetics								Thermodynamics
	R16 isolated domain		R16 in R1617^a		R17 isolated domain		R17 in R1617^b		Loss of stability of R16 domain (kcal·mol⁻¹)^c		Loss of stability of R17 domain (kcal·mol⁻¹)^c		Total loss in stability of R1617 on mutation (kcal·mol⁻¹)^c
	k_f (s⁻¹)	k_u (s⁻¹)	k_f (s⁻¹)	k_u (s⁻¹)	k_f (s⁻¹)	k_u (s⁻¹)	k_f (s⁻¹)	k_u (s⁻¹)	In R16 alone	In R1617	In R17 alone	In R1617	Total loss in stability of R1617 on mutation (kcal·mol⁻¹)^c
WT	130	0.0032	140	0.00090	27	0.00071	860	0.00012
Mutations in R16
I18V	66	0.019	52	0.0053	NA	NA	980	0.00021	1.4	1.6	NA	0.2	1.8
G101D	110	0.047	110	0.058	NA	NA	20	0.00032	1.7	2.6	NA	2.8	5.4
G101P	100	0.10	100	0.050	NA	NA	20	0.00036	2.2	2.6	NA	2.9	5.4
R103P	110	0.070	97	0.020	NA	NA	17	0.00034	1.9	2.0	NA	2.9	5.0
R103H	110	0.0064	150	0.0012	NA	NA	1300	0.00016	0.5	0.1	NA	−0.1	0.0
L104P	100	0.072	96	0.053	NA	NA	14	0.00041	2.0	2.6	NA	3.1	5.7
E105P	97	0.033	160	0.019	NA	NA	13	0.00043	1.5	1.7	NA	3.2	5.0
E106D	130	0.0053	110	0.00081	NA	NA	1000	0.000075	0.3	0	NA	−0.5	−0.4
Mutations in R17
Q5R	NA	NA	110	0.0013	18	0.00089	460	0.00019	NA	0.3	0.4	0.6	1.0
I18V	NA	NA	130	0.00077	7.6	0.0026	560	0.0011	NA	−0.1	1.5	1.6	1.5
N19S	NA	NA	120	0.0010	9.0	0.00080	810	0.00018	NA	0.1	0.7	0.3	0.4

	R15 isolated domain		R15 in R1516^d		R16 isolated domain		R16 in R1516^e		Loss of stability of R15 domain (kcal·mol⁻¹)^c		Loss of stability of R16 domain (kcal·mol⁻¹)^c		Total loss in stability of R1516 on mutation (kcal·mol⁻¹)^c
	k_f (s⁻¹)	k_u (s⁻¹)	k_f (s⁻¹)	k_u (s⁻¹)	k_f (s⁻¹)	k_u (s⁻¹)	k_f (s⁻¹)	k_u (s⁻¹)	In R15 alone	In R1516	In R16 alone	In R1516	Total loss in stability of R1516 on mutation (kcal·mol⁻¹)^c
WT	28 000	2.1	20 000	0.071	130	0.0032	730	0.00074
Mutations in R15
E106D	26 000	2.9	17 000	0.21	NA	NA	840	0.00092	0.2	0.7	NA	0.0	0.8
N105P	23 000	11	13 000	15	NA	NA	87	0.0033	1.1	3.4	NA	2.1	5.6

k_f and k_u are the rate constants for folding and unfolding, respectively, extrapolated to 0 m denaturant. The non-disease-related mutations are shown in bold and italics. The mutants I18V in R17 and N105P in R15 are discussed in detail in the text and are highlighted in red and blue, respectively.

As R16 folds first and unfolds last, the R16 kinetic parameters are always determined in the presence of an unfolded R17 neighbour.

As R16 folds first and unfolds last, the R17 kinetic parameters are always determined in the presence of a folded R16 neighbour.

The stability changes are calculated using the kinetic data presented, using the relationship ΔG_D-N = −RT ln(k_u/k_f). For the single domains, all values are very close to those determined by equilibrium measurements (as reported in Table 1), except for G101P. The kinetic measurements are more subject to experimental error because of the extrapolation of the unfolding data to 0 m denaturant. The errors for these estimates of ΔG_D-N are generally in the order of 0.1–0.3 kcal·mol⁻¹.

As R15 folds first and unfolds last, the R15 kinetic parameters are always determined in the presence of an unfolded R16 neighbour.

As R15 folds first and unfolds last, the R16 kinetic parameters are always determined in the presence of a folded R15 neighbour.