Table 2.
Specificity of PPKID and control peptidesa
| Peptides | Kd HisKIX (μM) |
Kd CA (μM) (Krel) |
Kd CalM (μM) (Krel) |
|
|---|---|---|---|---|
| PPKID4P | GPSQPTYPGDDAPVRRLSFFYILLDLYLDAPGVC | 0.515 ± 0.044 | 106 ± 12 (205) |
52 ± 12 (100) |
| PPKID6U | GISWPTFEGDDAPVRRLSFFYILLDLYLDAPGVC | 1.5 ± 0.1 | 79 ± 13 (53) |
> 168 (> 112) |
| CP | RRLSFFYILLDLYLDAPGVC | 2.4 ± 0.2 | 97 ± 6 (40) |
178 ± 42 (74) |
| CU | RRLSFFYILLDLYLDAPGVC | 21.5 ± 2.6 | 66 ± 11 (3) |
N.D. |
Each peptide was labeled on the C-terminal Cys residue with acetamidofluorescein for use in fluorescence polarization experiments. Kd values were determined by converting polarization data from two to three independent samples to fraction of fluorescently-labeled peptide bound values, which were fit to equilibrium binding equation (2). Residues selected at randomized positions are in red. P indicates a phosphopeptide. U indicates an unphosphorylated peptide. The phosphoserine residue in phosphopeptides is in bold. CA indicates that carbonic anhydrase II was used as the target protein; CalM indicates calmodulin was used as the target protein. The specificity ratio Krel is defined as Krel = Kd (CA or CalM) / Kd (HisKIX). N.D. indicates that the value was not determined.