Table 4. Kinetic constants (means±S.E.M.) measured by SPR for complex formation of chLBD mutants with immobilized oLep.
The S.E.M. values for the kon and koff values were calculated using the values obtained from their respective calculations in four or five concentrations of chLBD or chLBD mutants in each experiment. The numbering of amino acids is that of chLBD and the numbers in parentheses indicate the numbering in the full-size receptor (see also Figure 7).
| Mutant | kon (mol−1·s−1×105) | koff (s−1×10−3) | Kd (M×10−8) | χ2 |
|---|---|---|---|---|
| Wild-type* | 2.65±0.55 | 4.58±0.12 | 1.72±0.38 | 1.57–5.31 |
| Mutations aimed at humanizing the chLBD | ||||
| V103L (531)† | 2.94±0.43 | 6.65±0.67 | 2.26 | 0.8–2.97 |
| A105D (533) | 1.38±0.33 | 10.4±0.98 | 7.54 | 0.78 |
| D106S (534)† | 4.27±0.65 | 7.94±0.89 | 1.86 | 1.14–6.9 |
| D140N (568) | 1.50±0.22 | 5.19±0.63 | 3.34 | 1.88 |
| K142Q (570) | 1.85±0.28 | 6.67±0.86 | 3.59 | 2.78 |
| L162Y (590) | 2.07±0.46 | 5.54±0.66 | 2.66 | 1.88 |
| Mutations aimed at decreasing the interaction of chLBD with leptin | ||||
| Y14A (442) | 1.75±0.23 | 14.0±1.73‡ | 8.41 | 3.31 |
| Y14A/F73A (442/551) | No binding | |||
| R41A (469) | 1.05±0.33 | 64.0±9.55 | 63.8 | 0.42 |
| S42A (470) | 3.14±0.54 | 4.55±0.54 | 1.45 | 1.06 |
| K43A (471) | 2.08±0.32 | 7.35±0.80 | 3.53 | 1.06 |
| R41A/S42A/K43A (469–471) | 0.59±0.23 | 29.0±4.41 | 50.2 | 0.29 |
| F73A (501) | No binding | |||
| V76A/F77A (504/505) | No binding | |||
| L78A/L79A (506/507) | No binding | |||
| V76A/F77A/L78A/L79A (504–507) | No binding | |||
| V103A (531) | 0.42±0.16 | 10.80±1.93 | 25.7 | 1.24 |
| A105D/D106V (533/5343) | No binding | |||
| V135A (563)† | 4.59±0.66 | 5.55±0.73 | 1.21 | 1.32–4.52 |
| F136A (564) | 1.95±0.23 | 59.0±7.88 | 30.3 | 0.42 |
| V135A/F136A (563/564) | 3.48±0.49 | 87.0±9.97 | 28.7 | 0.48 |
| L191A (619)* | 3.93±0.70 | 5.78±0.67 | 1.47 | 1.97–4.40 |
* The results are means for three experiments. The respective S.E.M. values for kon, koff and Kd were 0.55, 0.12 and 0.38. Lower χ2 values indicate better fit.
† Average of two experiments.
‡ Values that differ by >3-fold from the WT LBD are shown in boldface.