Table 2.
Results from peptide binding studies
| Construct | Peptide | N1 | KD1 μM | ΔH1 (kcal/mol) | –T · ΔS1 (kcal/mol) | N2 | KD2 μM | ΔH2 (kcal/mol) | –T · ΔS2 (kcal/mol) | Comments |
|---|---|---|---|---|---|---|---|---|---|---|
| TtSlyDFL | S2 | 0.92 ± 0.02 | 0.161 ± 0.020 | –15.8 ± 0.11 | 6.6 | 1.00 ± 0.01 | 2.97 ± 0.22 | –9.4 ± 0.36 | 1.8 | 25 °C |
| S2 | 0.88 ± 0.02 | 0.128 ± 0.013 | –16.6 ± 0.10 | 6.1 | 0.92 ± 0.01 | 2.23 ± 0.15 | –10.5 ± 0.50 | 2.9 | 25 °C, tag-free | |
| S2 | 0.98 ± 0.01 | 0.150 ± 0.021 | –14.8 ± 0.11 | 5.5 | 1.05 ± 0.01 | 3.16 ± 0.18 | –8.9 ± 0.25 | 1.4 | 25 °C, 5 % DMSO | |
| S2 | 0.96 ± 0.03 | 0.113 ± 0.012 | –10.0 ± 0.11 | 0.7 | 0.96 ± 0.02 | 2.93 ± 0.12 | –4.9 ± 0.22 | –2.5 | 20 °C | |
| S3 | 0.89 ± 0.07 | 0.869 ± 0.156 | –9.1 ± 0.05 | 0.8 | 0.86 ± 0.04 | 22.94 ± 1.27 | –9.8 ± 0.39 | 3.5 | 25 °C | |
| T1 | 1.13 ± 0.05 | 158 ± 19 | –14.6 ± 0.80 | 9.5 | 25 °C | |||||
| SlpA linker | 1.44 ± 0.08 | 133 ± 23 | –0.4 ± 0.05 | –4.9 | 25 °C | |||||
| S2-minus1 | 0.89 ± 0.02 | 0.667 ± 0.059 | –16.6 ± 0.12 | 8.2 | 0.82 ± 0.02 | 7.25 ± 0.15 | –11.1 ± 0.29 | 4.1 | 25 °C | |
| S2-minus2 | 0.96 ± 0.03 | 1.472 ± 0.133 | –16.1 ± 0.21 | 8.1 | 0.99 ± 0.06 | 14.77 ± 1.09 | –4.3 ± 0.92 | –2.3 | 25 °C | |
| S2-minus3 | 0.94 ± 0.06 | 2.383 ± 0.321 | –17.0 ± 0.73 | 9.4 | 1.17 ± 0.06 | 9.35 ± 0.49 | –2.1 ± 1.41 | –4.8 | 25 °C | |
| S2-plus1 | 1.06 ± 0.03 | 0.317 ± 0.037 | –11.4 ± 0.13 | 2.3 | 0.94 ± 0.03 | 3.28 ± 0.08 | –7.8 ± 0.23 | 0.4 | 25 °C | |
| S2-plus2 | 0.88 ± 0.02 | 0.183 ± 0.018 | –13.1 ± 0.14 | 3.9 | 0.75 ± 0.04 | 5.68 ± 0.27 | –8.4 ± 0.39 | 1.2 | 25 °C | |
| S2-long2 | 0.72 ± 0.01 | 0.077 ± 0.007 | –10.8 ± 0.05 | 1.2 | 1.00 ± 0.01 | 6.62 ± 0.39 | –5.5 ± 0.14 | –1.4 | 20 °C | |
| S2-long4 | 0.74 ± 0.01 | 0.070 ± 0.004 | –11.1 ± 0.04 | 1.5 | 0.87 ± 0.01 | 4.16 ± 0.20 | –5.3 ± 0.10 | –1.9 | 20 °C | |
| S2-long6 | 0.74 ± 0.01 | 0.035 ± 0.003 | –10.5 ± 0.04 | 0.6 | 0.89 ± 0.01 | 4.52 ± 0.32 | –4.7 ± 0.11 | –2.5 | 20 °C | |
| S2-long8 | 0.95 ± 0.01 | 0.052 ± 0.006 | –11.7 ± 0.03 | 1.9 | 0.71 ± 0.02 | 7.93 ± 0.46 | –6.9 ± 0.22 | –0.1 | 20 °C | |
| S2-short2 | 0.72 ± 0.03 | 0.232 ± 0.043 | –9.7 ± 0.21 | 0.9 | 0.91 ± 0.02 | 3.16 ± 0.24 | –5.5 ± 0.34 | –1.9 | 20 °C | |
| S2-short4 | 0.73 ± 0.24 | 1.193 ± 0.666 | –10.7 ± 2.33 | 2.7 | 0.87 ± 0.23 | 4.24 ± 0.50 | –6.3 ± 3.02 | –0.9 | 20 °C | |
| S2-short6 | 1.80 ± 0.01 | 21.93 ± 0.51 | –5.4 ± 0.04 | –0.8 | 20 °C | |||||
| S2-short8 | 1.29 ± 0.30 | 163.4 ± 26.4 | –5.1 ± 1.54 | –0.1 | 20 °C | |||||
| S2-P25A | 0.92 ± 0.01 | 0.129 ± 0.028 | –16.6 ± 0.16 | 7.2 | 1.02 ± 0.04 | 7.04 ± 1.27 | –8.5 ± 0.82 | 1.5 | 25 °C | |
| S2-P29E | 0.88 ± 0.02 | 0.513 ± 0.052 | –15.8 ± 0.16 | 7.2 | 1.02 ± 0.04 | 5.68 ± 0.57 | –2.7 ± 0.48 | –4.5 | 25 °C | |
| S2-P25A/P29E | 1.13 ± 0.03 | 1.447 ± 0.295 | –14.1 ± 0.33 | 6.1 | 1.09 ± 0.24 | 44.05 ± 8.42 | –5.9 ± 1.91 | –0.1 | 25 °C, 5 % DMSO | |
| S2-P25N/P29N | 0.93 ± 0.02 | 0.234 ± 0.043 | –16.8 ± 0.12 | 7.8 | 1.18 ± 0.18 | 8.33 ± 1.54 | –2.3 ± 0.33 | –4.4 | 25 °C, 5 % DMSO | |
| SlpA linker P5T | 1.76 ± 0.12 | 130 ± 20 | –0.9 ± 0.10 | –4.4 | 25 °C | |||||
| S2-W23A | 0.76 ± 0.03 | 0.855 ± 0.332 | –15.8 ± 0.24 | 7.5 | 0.91 ± 0.40 | 18.83 ± 6.2 | –2.3 ± 0.97 | –4.2 | 25 °C | |
| TtSlyDΔIF | S2 | 0.91 ± 0.01 | 14.43 ± 0.46 | –10.0 ± 0.07 | 3.4 | 25 °C | ||||
| S2 | 0.88 ± 0.02 | 12.23 ± 0.24 | –5.9 ± 0.11 | 0.4 | 20 °C | |||||
| S3 | 0.89 ± 0.07 | 34.25 ± 3.40 | –10.5 ± 1.02 | 4.4 | 25 °C | |||||
| S2-P25A | 0.91 ± 0.01 | 12.13 ± 0.76 | –11.7 ± 0.24 | 5.0 | 25 °C | |||||
| S2-P29E | 0.69 ± 0.03 | 13.81 ± 0.88 | –10.8 ± 0.27 | 4.2 | 25 °C | |||||
| S2-P25A/P29E | 0.84 ± 0.06 | 28.98 ± 4.62 | –6.3 ± 0.58 | 0.1 | 25 °C, 5 % DMSO | |||||
| S2-W23A | 0.77 ± 0.02 | 17.06 ± 1.13 | –8.1 ± 0.25 | 1.6 | 25 °C |
In most cases, the Isothermal titration calorimetry data clearly supported the presence of two binding sites. Where data could be described by only a single binding site, the results are given in the columns for binding site 2. Note that the binding experiments with the proline double mutant peptides were carried out in 5 % dimethyl sulfoxide (DMSO) due to solubility issues. However, this is not expected to appreciably affect the experiments, as the S2 peptide was found to bind with similar affinities in both the presence and absence of 5 % DMSO