TABLE 3.
Biophysical properties of co-expressed IN·LEDGF heteromers
NA means not attempted.
| Protein | SECa | MALSb | IN:LEDGFd | Sedimentation velocity | f/f0 | Siegel and Monty | DLSb |
|---|---|---|---|---|---|---|---|
| Rs (Å) | Molar massc | s20,w | Molar massc | Rh (Å) | |||
| LEDGF(Cterm) | 35.4 ± 0.1 | 29,390 ± 1,910 (23,452) | NA | 1.7 | 1.8 | 24,298 | NA |
| IN(NTD-CCD)(F185K) LEDGF(IBD) | 38.5 ± 1.2 | 73,213 ± 4,838e (75,947) | 2:2 | 4.3 | 1.8 | 80,802 | NA |
| IN(tetra)-LEDGF(IBD) | 54.9 ± 1.8 | 159,720 ± 7,456 (157,721) | 4:2 | 7.2 | 1.8 | 160,165 | NA |
| IN-LEDGF(Cterm) | 73.7 ± 2.1 | 217,080 ± 16,194 (221,251) | 4:4 | 7.4 | 1.6 | 229,170 | 58.0 ± 3.8 |
| IN(tetra) LEDGF(Cterm) | 76.7 ± 1.6 | NA | NA | 7.7 | 1.6 | 243,655 | NA |
| IN(tetra)(D166N) LEDGF(Cterm) | NA | 209,720 ± 9049 (221,251) | 4:4 | NA | NA | NA | 57.1 ± 2.8 |
a The results presented are the average of 2–4 replicates. SEC measurements were made at room temperature.
b The results presented are the average of 3–5 replicates. DLS measurements were performed at 4 °C.
c Molar masses are presented in units of daltons. In parentheses, theoretical values as computed from primary sequence using SEDNTERP are presented.
d Stoichiometry was inferred from experimentally determined molecular mass; theoretical values are in parentheses.
e This preparation shows evidence of dissociation on silica-based resins not seen in other gel filtration media. In contrast to the other SEC-MALS analysis, this sample was analyzed using a Superdex 200 column in-line with MALS, using the theoretical extinction coefficient in lieu of its refractive index for concentration determination.