Table 3.
Thermodynamics of Splicing Factor Complexes Binding B3P3-Splice Site RNA
| Interaction:a | KD (nM) | ΔGb (kcal mol−1) | ΔH (kcal mol−1) | −TΔSc (kcal mol−1) | nd |
|---|---|---|---|---|---|
| B3P3 RNA Titrated into Splicing-Factor Complexes | |||||
| SF11–255-U2AF65R12U, site 1 | 2 ± 1 | −12.1 ± 0.1 | −60.1 ± 0.1 | 47.9 ± 0.1 | 0.8 ± 0.1 |
| SF11–255-U2AF65R12U, site 2 | 500 ± 10 | −8.7 ± 0.1 | −65.3 ± 0.1 | 56.6 ± 0.1 | 0.3 ± 0.1 |
| Phospho-SF11–255-U2AF65R12U, site 1 | 7 ± 5 | −11.5 ± 0.1 | −60.4 ± 0.1 | 48.9 ± 0.1 | 0.8 ± 0.1 |
| Phospho-SF11–255-U2AF65R12U, site 2 | 600 ± 80 | −8.7 ± 0.1 | −65.3 ± 0.1 | 57.6 ± 0.1 | 0.4 ± 0.1 |
| SF1pa-U2AF65R12U | 85 ± 1 | −9.8 ± 0.1 | −67.5 ± 0.1 | 57.7 ± 0.1 | 0.9 ± 0.1 |
| SF1mut-U2AF65R12U | 82 ± 1 | −9.8 ± 0.1 | −76.7 ± 0.1 | 66.9 ± 0.1 | 0.9 ± 0.1 |
| SF1mut-U2AF65R12Ue | 104 ± 7 | −9.7 ± 0.2 | −68.4 ± 0.2 | 58.6 ± 0.2 | 1.0 ± 0.1 |
| Splicing-Factor Complexes Titrated into B3P3 RNAf | |||||
| SF11–255-U2AF65R12U, site 1 (reverse) | 160 ± 80 | −9.5 ± 0.1 | −22.3 ± 0.1 | 12.8 ± 0.1 | 0.6 ± 0.1 |
| SF11–255-U2AF65R12U, site 2 (reverse) | 7 ± 1 | −11.3 ± 0.1 | −53.2 ± 0.1 | 42.0 ± 0.1 | 0.9 ± 0.2 |
| Phospho-SF11–255-U2AF65R12U, site 2 (reverse) | 115 ± 5 | −9.8 ± 0.1 | −81.1 ± 4.9 | 71.4 ± 4.9 | 0.5 ± 0.1 |
| Phospho-SF11–255-U2AF65R12U, site 1 (reverse) | 18 ± 3 | −10.8 ± 0.1 | −36.2 ± 4.5 | 25.5 ± 4.7 | 0.7 ± 0.2 |
a
Values are expressed as the mean ± SD of at least two independent titrations.
b
ΔG values were calculated using the equation ΔG = −RT ln (KA) where T=303 K
c
−TΔS values were calculated using the equation −TΔS = ΔG − ΔH.
d
Fits are “ligand-in-cell” such that “n” is the apparent stoichiometry of protein/RNA.
e
Dialyzed into 100 mM NaCl and 25 mM sodium phosphate buffer (pH 7.4). Macromolecules for other experiments in this table were in 100 mM NaCl, 25 mM HEPES (pH 7.4).
f
Apparent stoichiometry of protein/RNA (n).