Table 2.
Effect of Ligands on the Apparent Affinity of the Labeled Peptides Used in This Study
| (Putative) β-strand | α-Helix | |||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | Kd μM) | −ΔG kcal/mol | ||
| NCoR | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | −L | 0.6 ± 0.2 | 8.5 ± 0.2 | ||
| ID1B-2 | ||||||||||||||||||||||||
| FITC | ||||||||||||||||||||||||
| NCoR | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | Heme 5 µM | 8.3 ± 1.4 | 6.8 ± 0.1 | ||
| ID1B-2 | ||||||||||||||||||||||||
| FITC | ||||||||||||||||||||||||
| NCoR | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | Heme 10 µM | 13.7 ± 4.4 | 6.6 ± 0.2 | ||
| ID1B-2 | ||||||||||||||||||||||||
| FITC | ||||||||||||||||||||||||
| NCoR | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | SGN 100 µM | 0.1 ± 0.04 | 9.3 ± 0.2 | ||
| ID1B-2 | ||||||||||||||||||||||||
| FITC | ||||||||||||||||||||||||
| NCoR | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | SGN 20 µM | 0.08 ± 0.03 | 9.6 ± 0.2 | ||
| ID1B-2 | ||||||||||||||||||||||||
| FITC | ||||||||||||||||||||||||
| NCoR | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | SD7 100 µM | 3.8 ± 2.3 | 7.4 ± 0.3 | ||
| ID1B-2 | ||||||||||||||||||||||||
| FITC | ||||||||||||||||||||||||
| NCoR | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | SD7 20 µM | 1.8 ± 0.5 | 7.7 ± 0.1 | ||
| ID1B-2 | ||||||||||||||||||||||||
| FITC | ||||||||||||||||||||||||
| Smrt | R | V | V | T | L | A | Q | H | I | S | E | V | I | T | Q | D | Y | T | R | -L | 0.2 ± 0.1 | 9.1 ± 0.2 | ||
| ID1B-2 | ||||||||||||||||||||||||
| Rho | ||||||||||||||||||||||||
| Smrt | R | V | V | T | L | A | Q | H | I | S | E | V | I | T | Q | D | Y | T | R | Heme 5 µM | 4.2 ± 0.7 | 7.2 ± 0.1 | ||
| ID1B-2 | ||||||||||||||||||||||||
| Rho | ||||||||||||||||||||||||
| Smrt | R | V | V | T | L | A | Q | H | I | S | E | V | I | T | Q | D | Y | T | R | Heme 10 µM | 6.3 ± 1.02 | 7.01 ± 0.1 | ||
| ID1B-2 | ||||||||||||||||||||||||
| Rho | ||||||||||||||||||||||||
| Smrt | R | V | V | T | L | A | Q | H | I | S | E | V | I | T | Q | D | Y | T | R | SGN 100 µM | 0.08 ± 0.01 | 9.5 ± 0.07 | ||
| ID1B-2 | ||||||||||||||||||||||||
| Rho | ||||||||||||||||||||||||
| Smrt | R | V | V | T | L | A | Q | H | I | S | E | V | I | T | Q | D | Y | T | R | SD7 100 µM | 0.9 ± 0.5 | 8.2 ± 0.35 | ||
| ID1B-2 | ||||||||||||||||||||||||
| Rho | ||||||||||||||||||||||||
| NCoR5- | T | H | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | −L | 0.35 ± 0.1 | 8.7 ± 0.2 |
| Atto | S | F | A | D | P | A | S | N | L | G | L | E | D | I | I | R | K | A | L | M | G | |||
| NCoR5 | T | H | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | −L | 0.5 ± 0 | 8.4 ± 0 |
| S | F | A | D | P | A | S | N | L | G | L | E | D | I | I | R | K | A | L | M | G | ||||
| NCoR5- | T | H | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | Heme 5 µM | 46 ± 30 | 5.9 ± 0.4 |
| Atto | S | F | A | D | P | A | S | N | L | G | L | E | D | I | I | R | K | A | L | M | G | |||
| NCoR5- | T | H | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | SGN 100 µM | 0.1 ± 0 | 9.4 ± 0 |
| Atto | S | F | A | D | P | A | S | N | L | G | L | E | D | I | I | R | K | A | L | M | G | |||
| NCoR5- | T | H | R | L | I | T | L | A | D | H | I | C | Q | I | I | T | Q | D | F | A | R | SD7 100 µM | 1.4 ± 0.4 | 7.9 ± 0.2 |
| Atto | S | F | A | D | P | A | S | N | L | G | L | E | D | I | I | R | K | A | L | M | G | |||
Estimated errors are standard deviations from three or more experiments. Labeled peptides were used at a concentration of 4 nM. Estimated errors are standard deviations from 3 or more experiments. The label –L indicates in the absence of ligand. The sequences ID1 and ID2 motifs of the NCoR5 construct are given here. The complete 266 residue sequence can be found in the Materials and Methods section. The secondary structures of the corresponding residues in the crystal structures are indicated in yellow for the putative β-strand in yellow and the α-helix in green in the top line of the table. Red indicates that the residues at these positions have been deleted for the corresponding peptide. FITC, Rho, and Atto refer to N-terminal labeling of the peptides with either fluorescein, rhodamine 110 and Atto647N.