Table 1.
Structural and functional properties for wild-type and HE/HPP α0-1 mutants
| α0-1 | Tm,* °C | Kd,† μM | n† | ΔH,† kcal/mol | −TΔS,‡ kcal/mol | ΔG,‡ kcal/mol | Affinity category§ |
|---|---|---|---|---|---|---|---|
| Wild-type | 54.6 | 0.4 ± 0.1 | 0.9 ± 0.06 | −33.1 ± 3.5 | 24.5 ± 3.4 | −8.6 | wt |
| I24S | 53.2 | >100 | − | − | − | − | 3 |
| I24T | 54.4 | 41 ± 7 | 0.9 ± 0.06 | −29.0 ± 7.9 | 23.2 ± 8.0 | −5.8 | 2 |
| R28C | 55.6 | >200 | − | − | − | − | 3 |
| R28H | 53.6 | >200 | − | − | − | − | 3 |
| R28L | 53.1 | 67 ± 4 | 1.1 ± 0.03 | −21.2 ± 1.4 | 15.6 ± 1.5 | −5.6 | 2 |
| R28S | 53.3 | >200 | − | − | − | − | 3 |
| V31A | 54.5 | 23 ± 3 | 1.0 ± 0.06 | −32.1 ± 6.5 | 26.0 ± 6.6 | −6.1 | 2 |
| R34W | 49.1 | 0.1 ± 0.0 | 0.9 ± 0.02 | −40.5 ± 3.6 | 31.1 ± 3.6 | −9.4 | ∼wt |
| R41W | 53.4 | 8.7 ± 0.4 | 1.0 ± 0.05 | −29.4 ± 4.7 | 22.6 ± 4.8 | −6.8 | 1 |
| R45S | 55.1 | >200 | − | − | − | − | 3 |
| R45T | 54.6 | 14 ± 2.0 | 0.9 ± 0.01 | −29.5 ± 1.0 | 23.1 ± 1.1 | −6.4 | 1 |
| G46V | 52.8 | 3.7 ± 0.7 | 0.9 ± 0.02 | −31.3 ± 2.3 | 24.1 ± 2.4 | −7.2 | 1 |
| K48R | 54.7 | 0.5 ± 0.1 | 0.9 ± 0.05 | −33.5 ± 0.4 | 25.0 ± 3.9 | −8.5 | ∼wt |
| L49F | 55.4 | 30 ± 1 | 1.0 ± 0.03 | −14.9 ± 1.2 | 8.9 ± 1.2 | −6.0 | 2 |
Values are from single DSC experiments.
Averages and standard deviations for binding to wild-type β16-17 in replicate ITC experiments (triplicates for all mutants and four replicates for wild-type). All experiments shown here were conducted at 23° C (see text for wild-type values at other temperatures). Average protein concentrations for wild-type β16-17 (cell) and α0-1 constructs (syringe) are, respectively: 19 μM and 563 μM for WT, 24 μM and 565 μM for I24S, 30 μM and 653 μM for I24T, 35 μM and 511 μM for R28C, 33 μM and 603 μM for R28H, 28 μM and 510 μM for R28L, 38 μM and 820 μM for R28S, 24 μM and 575 μM V31A, 14 μM and 270 μM for R34W, 29 μM and 674 μM for R41W, 28 μM and 560 μM for R45S, 29 μM and 664 μM for R45T, 29 μM and 619 μM for G46V, 25 μM and 576 μM for K48R, 32 μM and 541 μM for L49F. For experiments where binding was below quantifiable limits, upper limits for Kd were estimated using Origin software for the protein concentrations used in the analysis; these experiments were performed in duplicate.
Values for −TΔS and ΔG are calculated from the experimentally determined association constant (K) and enthalpy (ΔH) using the relationships: RTlnK = ΔG = ΔH-TΔS where ΔG is the observed free energy, R is the gas constant, T is the experimental temperature in Kelvin, and ΔS is the calculated entropy.
Binding affinity groups were defined as follows: wt indicates similar to wild-type; 1, approximately 10-fold weaker than wild-type; 2, approximately 100-fold weaker than wild-type; 3, marginal or no detectable binding.