Table 1. Urea-induced unfolding parameters for variants that contain alanine to glycine substitutions in Nank1-5, Nank1-5C1, and Nank1-5C2.
| ΔG°H2O (kcal•mol-1) | m-value (kcal•mol-1•M-1) | Cm (M) | ||||
|---|---|---|---|---|---|---|
| CD | Fluorescence | CD | Fluorescence | CD | Fluorescence | |
| Nank1-5 | 3.21 ± 0.09 | 3.29 ± 0.03 | 1.78 ± 0.03 | 1.84 ± 0.01 | 1.80 ± 0.09 | 1.78 ± 0.02 |
|
| ||||||
| Nank1-5 (AG1) | 3.06 ± 0.06 | 3.17 ± 0.13 | 1.83 ± 0.02 | 1.89 ± 0.05 | 1.67 ± 0.02 | 1.68 ± 0.03 |
| Nank1-5C1 | 7.31 ± 0.17 | 7.50 ± 0.09 | 1.98 ± 0.10 | 2.05 ± 0.03 | 3.67 ± 0.04 | 3.65 ± 0.02 |
|
| ||||||
| Nank1-5C1 (AG1) | 8.21 ± 0.06 | 8.16 ± 0.24 | 2.25 ± 0.01 | 2.22 ± 0.07 | 3.65 ± 0.01 | 3.67 ± 0.01 |
| Nank1-5C1 (AG2) | 3.72 ± 0.09 | 3.88 ± 0.09 | 1.77 ± 0.04 | 1.85 ± 0.05 | 2.10 ± 0.07 | 2.10 ± 0.07 |
| Nank1-5C1 (AG3) | 3.61 ± 0.13 | 4.00 ± 0.16 | 1.84 ± 0.03 | 2.01 ± 0.07 | 1.96 ± 0.04 | 1.99 ± 0.01 |
| Nank1-5C1 (AG4) | 5.52 ± 0.04 | 5.69 ± 0.11 | 2.32 ± 0.01 | 2.41 ± 0.04 | 2.38 ± 0.01 | 2.36 ± 0.01 |
| Nank1-5C1 (AG5) | 5.71 ± 0.09 | 5.77 ± 0.07 | 2.22 ± 0.03 | 2.26 ± 0.02 | 2.58 ± 0.01 | 2.56 ± 0.01 |
| Nank1-5C1 (AGC1) | 5.68 ± 0.06 | 5.66 ± 0.08 | 2.18 ± 0.01 | 2.17 ± 0.02 | 2.61 ± 0.01 | 2.61 ± 0.02 |
| Nank1-5C2 | 13.0 ± 0.03 | 13.25 ± 0.40 | 2.28 ± 0.01 | 2.30 ± 0.07 | 5.70 ± 0.01 | 5.70 ± 0.01 |
|
| ||||||
| Nank1-5C2 (AG1) | 12.8 ± 0.12 | 12.62 ± 0.09 | 2.21 ± 0.02 | 2.17 ± 0.01 | 5.77 ± 0.02 | 5.82 ± 0.01 |
| Nank1-5C2 (AG2) | NAa | (8.98, 7.95) | NAa | (1.78, 1.59) | NAa | (5.06, 4.98) |
| Nank1-5C2 (AG3) | NAa | 8.12 ± 0.22 | NAa | 1.64 ± 0.04 | NAa | 4.97 ± 0.01 |
| Nank1-5C2 (AG4) | 10.1 ± 0.05 | 9.59 ± 0.10 | 2.14 ± 0.01 | 2.06 ± 0.03 | 4.72 ± 0.02 | 4.66 ± 0.02 |
| Nank1-5C2 (AG5) | 11.9 ± 0.07 | 11.46 ± 0.28 | 2.44 ± 0.02 | 2.35 ± 0.05 | 4.89 ± 0.01 | 4.88 ± 0.01 |
| Nank1-5C2 (AGC1) | 11.8 ± 0.08 | 11.65 ± 0.13 | 2.45 ± 0.02 | 2.41 ± 0.03 | 4.85 ± 0.01 | 4.84 ± 0.01 |
| Nank1-5C2 (AGC2) | 11.7 ± 0.04 | 11.61 ± 0.14 | 2.50 ± 0.01 | 2.48 ± 0.03 | 4.69 ± 0.01 | 4.68 ± 0.01 |
Parameters were estimated by fitting urea-induced unfolding transitions using nonlinear least-squares as described.42 Uncertainties are standard errors on the mean of at least three titrations.
a
Unfolding parameters determined using CD could not be obtained for Nank1-5C2 (AG2) and Nank1-5C2 (AG3) due to the existence of a folding intermediate. Conditions: 25 mM Tris•HCl (pH 8.0), 150 mM NaCl, 20 °C.