Table 1. Binding Constant Summary of L3MBTL1206–519 and Its Gating Loop Mutants.
| Protein | WT | D355N | D355A | N358Q | N358A |
|---|---|---|---|---|---|
| H1.418–32 | H2N-TPVKKKARK26SAGAAK-COOH | ||||
| H1.4unmod | >500 | >500 | >1000 | >1000 | >500 |
| H1.4K26me1 | 12 ± 1 | 67 ± 7 | >500 | 77 ± 10 | 63 ± 4 |
| H1.4K26me2 | 15 ± 2 | 47 ± 8 | >500 | 44 ± 6 | 39 ± 6 |
| H1.4K26me3 | 200 ± 41 | 290 ± 53 | >1000 | >500 | 230 ± 34 |
| H31–15 | H2N-ARTK4QTARK9STGGKAY-COOH | ||||
| H3unmod | >500 | >1000 | >1000 | >1000 | >1000 |
| H3K4me1 | 6 ± 1 | 78 ± 12 | >400 | 30 ± 5 | 66 ± 5 |
| H3K4me2 | 75 ± 12 | 142 ± 13 | >400 | 120 ± 19 | 145 ± 9 |
| H3K4me3 | >500 | 350 ± 23 | >1000 | 200 ±11 | 250 ± 24 |
| H3K9me1 | 10 ± 2 | 120 ± 24 | >500 | 63 ± 7 | 170 ± 11 |
| H3K9me2 | 20 ± 3 | 91 ± 19 | >500 | 72 ± 9 | 200 ±11 |
| H3K9me3 | 400 ± 68 | >500 | >1000 | 250 ±15 | >1000 |
| H3K9me1S10ph | 120 ± 19 | — | — | — | — |
| H3K9me3S10ph | >500 | — | — | — | — |
| H31–15scram | H2N-TAGASRKGKme1QRKTATY-COOH | ||||
| H3Kme1scram | 41 ± 5 | 266 ± 31 | >1000 | 71 ± 5 | 125 ± 13 |
| H319–35 | H2N-QLATKAARK27SAPATGGVY-COOH | ||||
| H3unmod | >1000 | >1000 | — | >1000 | — |
| H3K27me1 | 41 ± 7 | 500 ±121 | — | 110 ± 11 | — |
| H3K27me2 | 75 ± 19 | 500 ± 99 | — | 140 ± 13 | — |
| H3K27me3 | >500 | >1000 | — | >500 | — |
| H328–43 | H2N-SAPATGGVK36KPHRYRPY-COOH | ||||
| H3unmod | >700 | >1000 | >1000 | >1000 | >1000 |
| H3K36me1 | 16 ± 2 | 84 ± 10 | >500 | 83 ± 18 | 180 ± 15 |
| H3K36me2 | 27 ± 5 | 86 ± 18 | >500 | 85 ± 22 | 130 ± 28 |
| H3K36me3 | 400 ±115 | >500 | >500 | >500 | >500 |
| H412–27 | H2N-KGGAKRHRK20VLRDNIQ-COOH | ||||
| H4unmod | 410 ±131 | >500 | >500 | >500 | >500 |
| H4K20me1 | 5 ± 1 | 60 ± 9 | 420 ± 75 | 26 ± 6 | 100 ± 13 |
| H4K20me2 | 6 ± 2 | 43 ± 6 | 205 ±18 | 23 ± 5 | 50 ± 11 |
| H4K20me3 | 190 ± 31 | 290 ± 35 | 500 ± 67 | 160 ± 20 | 190 ± 23 |
| H4K16acK20me1 | 16 ± 1 | — | — | — | — |
| H4K16ac | >500 | — | — | — | — |
| H412–27scram | H2N-LNRQDIAGKme1GKHVKRR-COOH | ||||
| H4Kme1scram | 14 ± 2 | 165 ± 35 | >500 | 52 ± 10 | 150 ± 24 |
Binding constants were obtained from fluorescence polarization-based binding assay and shown at μM scale. Fluorescence anisotropy of the fluorescein-labeled peptide was used to determine the apparent dissociation constant KD (based on an assumption of a 1:1 binding stoichiometry). In each case, parameters are reported as the mean (±average deviation from the mean) obtained from three independent titration experiments. WT, wild type; —, not measured.