Table 3.
Effect of modifications on DNA bend angle in protein-DNA complexes
| Protein | DNAa | E (Free DNA)b,c,d | E (+EcoRV) | Rfree (Å)e | R+EcoRV (Å)e | Bend Angle (°)f |
|---|---|---|---|---|---|---|
| wild type | GATATC | 0.62 ±0.01 | 0.75 ±0.01 | 48.3 ±0.1 | 43.6 ±0.3 | 51 ±2 |
| wild type | PMe-5 | 0.63 ±0.02 | 0.76 ±0.02 | 48.1 ±0.6 | 43.4 ±0.6 | 51 ±4 |
| wild type | PMe-2 | 0.55 ±0.01 | 0.68 ±0.03 | 50.8 ±0.5 | 46.5 ±1.0 | 48 ±3 |
| wild type | PMe+3 | 0.51 ±0.03 | 0.64 ±0.04 | 52.0 ±1.0 | 47.8 ±1.3 | 47 ±2 |
| K119A | GATATC | 0.61 ±0.01 | 0.77 ±0.01 | 48.6 ±0.1 | 42.9 ±0.4 | 56 ±2 |
| K119A | PMe-2 | 0.56 ±0.01 | 0.76 ±0.05 | 50.6 ±0.6 | 43.3 ±1.9 | 56 ±8 |
| R221A | GATATC | 0.61 ±0.01 | 0.74 ±0.02 | 48.9 ±0.4 | 44.1 ±0.9 | 51 ±3 |
| R221A | PMe-2 | 0.54 ±0.01 | 0.62 ±0.03 | 51.5 ±0.5 | 48.4 ±0.5 | 39 ±3 |
| R226A | GATATC | 0.61 ±0.01 | 0.73 ±0.03 | 48.8 ±0.4 | 44.4 ±1.0 | 49 ±4 |
| R226A | PMe-5 | 0.63 ±0.02 | 0.74 ±0.04 | 48.2 ±0.6 | 44.0 ±1.3 | 48 ±5 |
| R140A | GATATC | 0.61 ±0.01 | 0.73 ±0.03 | 48.6 ±0.2 | 44.3 ±1.0 | 48 ±6 |
| R140A | PMe+3 | 0.51 ±0.02 | 0.63 ±0.03 | 52.2 ±0.6 | 48.0 ±1.0 | 46 ±3 |
The double-stranded 14 bp oligonucleotide d (AGAAGATATCTTGA) was used as the parent sequence context for all FRET experiments.
FRET efficiency (E) was measured in binding buffer plus 0.1 M K+ at 294K
Means and standard deviations are for at least 2 determinations of FRET efficiency.
FRET efficiency was calculated from the acceptor emission as described in Materials and Methods.
Interfluorophore distance (R) was calculated from the equation E = R06/(R06 + R6) where R0 is the Förster distance for the Fl/Ta pair (52.5Å).
Calculated from the equation; Bend Angle = 180-[2 (sin−1[(R+EcoRV/2)/(Rfree/2)]]; values were calculated from individual experiments before calculating mean and standard deviation.