Table 2.
Biophysical analysis of rHC1 dimerization
Radius of gyration (Rg), maximum dimension (Dmax), approximate molecular weight (Mwt), and sedimentation coefficient (s(20,w)) values were derived from SAXS and AUC data for WT and D298A rHC1. All D298A data and WT data collected in the presence of 2.5 mm EDTA are consistent with a monomeric state. WT rHC1 with 5 mm MgCl2 or 5 mm MnCl2 is dimeric. The data from “as purified” WT rHC1 and WT rHC1 in 5 mm CaCl2 are consistent with a mixture of monomer and dimer; this is presumably due to trace amounts of Mg2+ ions present in various buffer components. The AUC data are derived from equilibrium experiments performed in triplicate at three different speeds; SAXS data are from data processed by AUTORG and with DATGNOM (i.e. with no imposed constraints). The molecular weight of an rHC1 monomer from intact mass spectrometry is 73,802 Da.
| Protein | Metal ion | RgSAXS | DmaxSAXS | MwtSAXS (ratio SAXS mass/intact mass)a | s(20,w)AUC | s(20,w)SAXS |
|---|---|---|---|---|---|---|
| Å | Å | kDa | S | S | ||
| rHC1 WT | Noneb | 35.2 | 123 | |||
| EDTA (2.5 mm) | 31.8 | 112 | 78 (1.06) | 4.39 | 4.50 | |
| Mg2+ (5 mm) | 49.5 | 170 | 140 (1.89) | 4.59 (monomer), 6.11(dimer) | 6.24 | |
| Mn2+ (5 mm) | 51.2 | 172 | ||||
| Ca2+ (5 mm) | 38.5 | 135 | ||||
| rHC1 D298A | Noneb | 33.3 | 114 | |||
| Mg2+ (5 mm) | 33.3 | 114 | ||||
| Mn2+ (5 mm) | 32.8 | 105 | ||||
| Ca2+ (5 mm) | 32.8 | 115 | ||||
| IαI | Mg2+ (5 mm) | 49.2 | 170 |
a Calculated using the method in Ref. 83.
b As purified.