Table 1.
Fractional factorial folding screen
| No.a | Patternb | Conditions
|
Analysis
|
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Protein, mg/ml | pH | IS,c mM | Td | Divalentse | Polar additive | Nonpolar additive,f % | Chaotrope | Reduced/oxidizedg | Detergent,h mM | Ligand,i mM | PEG,j % | SDS/PAGEk | SEC,l % | Ligand bindingm | ||
| 1 | −+−++−+−−+−+ | 0.1 | 8.5 | 10 | 20 | Mg, Ca | None | 21 | None | DTT | 5.0 | None | 0.05 | − | 1.0 | 782 |
| 2 | +++−−−−−−−−+ | 1.0 | 8.5 | 250 | 4 | EDTA | None | None | None | DTT | None | None | 0.05 | ++ | 6.0 | 8318 |
| 3 | −+++−+−+−+−− | 0.1 | 8.5 | 250 | 20 | EDTA | 0.5 M Arg | None | 0.75 M Gdn⋅HCl | DTT | 5.0 | None | None | ++ | <0.1 | <200 |
| 4 | −+−−−+−++−++ | 0.1 | 8.5 | 10 | 4 | EDTA | 0.5 M Arg | None | 0.75 M Gdn⋅HCl | GSH:GSSG | None | 10.0 | 0.05 | + | <0.1 | <200 |
| 5 | +−+−++−−++−− | 1.0 | 6.0 | 250 | 4 | Mg, Ca | 0.5 M Arg | None | None | GSH:GSSG | 5.0 | None | None | − | 1.0 | <200 |
| 6 | +−++−−++−−+− | 1.0 | 6.0 | 250 | 20 | EDTA | None | 21 | 0.75 M Gdn⋅HCl | DTT | None | 10.0 | None | − | 0.4 | <200 |
| 7 | ++++++++++++ | 1.0 | 8.5 | 250 | 20 | Mg, Ca | 0.5 M Arg | 21 | 0.75 M Gdn⋅HCl | GSH:GSSG | 5.0 | 10.0 | 0.05 | − | 0.8 | <200 |
| 8 | −−−−+−−+−++− | 0.1 | 6.0 | 10 | 4 | Mg, Ca | None | None | 0.75 M Gdn⋅HCl | DTT | 5.0 | 10.0 | None | + | 4.0 | 2891 |
| 9 | +−−+++−−−−++ | 1.0 | 6.0 | 10 | 20 | Mg, Ca | 0.5 M Arg | None | None | DTT | None | 10.0 | 0.05 | − | 0.6 | <200 |
| 10 | −−+−−++−−+++ | 0.1 | 6.0 | 250 | 4 | EDTA | 0.5 M Arg | 21 | None | DTT | 5.0 | 10.0 | 0.05 | + | 3.0 | <200 |
| 11 | −−+++−−++−−+ | 0.1 | 6.0 | 250 | 20 | Mg, Ca | None | None | 0.75 M Gdn⋅HCl | GSH:GSSG | None | None | 0.05 | − | <0.1 | <200 |
| 12 | +−−−−−++++−+ | 1.0 | 6.0 | 10 | 4 | EDTA | None | 21 | 0.75 M Gdn⋅HCl | GSH:GSSG | 5.0 | None | 0.05 | − | 1.0 | <200 |
| 13 | ++−−++++−−−− | 1.0 | 8.5 | 10 | 4 | Mg, Ca | 0.5 M Arg | 21 | 0.75 M Gdn⋅HCl | DTT | None | None | None | ++ | 5.0 | 5490 |
| 14 | ++−+−−−−+++− | 1.0 | 8.5 | 10 | 20 | EDTA | None | None | None | GSH:GSSG | 5.0 | 10.0 | None | − | 0.3 | <200 |
| 15 | −++−+−+−+−+− | 0.1 | 8.5 | 250 | 4 | Mg, Ca | None | 21 | None | GSH:GSSG | None | 10.0 | None | ++ | 8.0 | 6183 |
| 16 | −−−+−++−+−−− | 0.1 | 6.0 | 10 | 20 | EDTA | 0.5 M Arg | 21 | None | GSH:GSSG | None | None | None | − | <0.1 | <200 |
Solution number.
Pattern of factor levels.
Ionic strength, molar ratio of NaCl to KCl was 25:1.
Temperature in °C.
MgCl2, CaCl2 concentrations were 2 mM; EDTA concentration was 1.0 mM.
The nonpolar additive was composed of 20% glycerol (v/v) and 1% sucrose (w/v).
Concentration of DTT was 1.0 mM, and the concentrations of reduced (GSH) and oxidized (GSSG) glutathione were 1.0 and 0.1 mM, respectively.
1,2-Diheptanoyl-sn-glycero-3-phorphocholine.
l-Glutamate.
PEG MWave = 3350 Da, and the concentration was w/v.
Protein concentration in the supernatant following dialysis against refolding buffers and centrifugation at 128,000 × g for 30 min (4°C) was estimated by SDS/PAGE. − corresponds to <0.1 μg or to <3% yield of water-soluble HS1S2; “+” and “++” correspond to ∼10 and ∼20% yields of water-soluble HS1S2, respectively.
Folding yield estimated by integration of monomer peak from SEC chromatogram.
The units are cpm. [3H]AMPA (20 nM, 10.6 Ci/mmol) and 2 or 20 μl of each refolding mixture with an initial protein concentration of 1.0 or 0.1 mg/ml, respectively, were used to measure specific ligand binding as described in Materials and Methods.