Having established the H31-27K9Me3, H31-27K18Ac, and H31-27K9Me3K18Ac peptides as binding partners for the TRIM33α and TRIM33β PHD-BRD, we next sought to investigate the contributions of key residues in the PHD and BRD to peptide recognition. To achieve this, we used the signal response curves for mutants of the TRIM24, TRIM33α, and TRIM33β PHD-BRD, and biotinylated peptides in the AlphaScreen assay. To probe binding to the PHD we made the W828A mutation in TRIM24, and the W889A mutation in TRIM33α and β. To probe BRD binding we made both the N980A and N980F mutants in TRIM24 and the N1039F mutation in TRIM33α and β. The N to F mutation has previously been shown to be more effective at preventing KAc binding to the BRD than the N to A mutation at the same position. 58 The W828A and W889A mutants generated no signal in the AlphaScreen assay when the proteins were incubated with varying concentrations of the H31-27K9Me3 peptide. This indicates that this mutation abolishes PHD-mediated recognition of KMe3 for all three proteins (Table ). This result confirms the importance of W828/W889 in the recognition of the H3K9Me3 modification. The affinity of the dual modified H31-27K9Me3K18Ac was also reduced by the W828A/W889A mutations, compared to the wild type proteins. The largest reduction in signal response for the H31-27K9Me3K18Ac peptide occurred for the TRIM33α PHD-BRD, suggesting that the K9Me3 modification provides a substantial contribution to the TRIM33α PHD-BRD affinity for the peptide, in line with the ITC data.