Table 1.
Physical properties of WT and mutant HypA proteins.
| HypA | Subunit MW (Da) | Oligomer MW (Da)b | Tm(°C) | Tm(°C) | Kd(ITC) | ΔH (ITC) of Ni binding (kcal/mol) | n (ITC) | Zn:apo-HypAe |
|---|---|---|---|---|---|---|---|---|
| Obsa (calc’d) | −Ni | +Ni | (μM) | (Ni/protein) | ||||
| WT | 13,203 (13,202) | 26,600 |
pH = 7.2 58(2)c 59.1(3)d |
pH = 7.2 70(5)c 70.5(4)d |
pH = 7.2 1.08(6) |
pH = 7.2 −18.9(1) |
pH = 7.2 1.050(5) |
pH = 7.2 0.94:1 |
|
pH = 6.3 60(4)d |
pH = 6.3 71(1)d |
pH = 6.3 17(2) |
pH = 6.3 −9.0(6) |
pH = 6.3 0.46(3) |
pH = 6.3 0.92:1 |
|||
|
pH = 8.3 3.7(3) |
pH = 8.3 −9.75(7) |
pH = 8.3 0.936(4) |
||||||
| C74A | 13,169 (13,170) | 28,000 | 63.5(1)d | 69.7(4)d | 54(4) | −13.8(3) | 0.506(8) | 1.03:1 |
| C77A | 13,171 (13,170) | 29,000 | 72.3(1)d | 66.8(5)d | 48(2) | −13.0(2) | 0.478(5) | 1.05:1 |
| C91A | 13,170 (13,170) | 28,300 | 57.6(6)d | 75.8(4)d | 22(3) | −8.6(1) | 0.44(6) | 0.92:1 |
| C94A | 13,171 (13,170) | 29,500 | 66.3(9)d | 73.3(1)d | 16(2) | −16(1) | 0.48(3) | 0.89:1 |
| C94D | 13,213 (13,214) | 73.9(2)d | 79.7(2)d | 22(2) | −22(1) | 0.46(2) | 0.93:1 | |
| H79A | 13,136 (13,136) | 27,000 | 67.1(2)d | 68.6(2)d |
pH = 7.2 3.2(3) |
pH = 7.2 −34.3(3) |
pH = 7.2 0.932(5) |
0.97:1 |
| H95A | 13,136 (13,136) | 30,000 | 66.8(2)d | 68.0(2)d |
pH = 7.2 2.1(1) |
pH = 7.2 −27.6(3) |
pH = 7.2 0.936(6) |
1.06:1 |
|
pH = 6.3 21(2) |
pH = 6.3 −45.7(2) |
pH = 6.3 0.96(3) |
a
Determined by ESI-MS;
b
Determined by size-exclusion chromatography;
c
Melting temperature determined by DSC;
d
Melting temperature determined by performing a thermal melt and monitoring the CD signature at 222 nm.
e
Determined using ICP-OES for [Zn] and optical spectrum calibrated with amino acid analysis for [protein].