Fig. 5: Identification of photocrosslinking site.
From: Identification of photocrosslinking peptide ligands by mRNA display
a Confirmation of photocrosslinking of BRD3-BD2 by intact mass spectrometry. A mixture of BRD3-BD2 (2 µM) and cyclic peptides (20 µM) were exposed to UV irradiation for 30 min and analysed by LC–MS. The mass shifts correspond to adducts formed from the addition of one molecule of Bpa-P1 or Bpa-P2. b, Ribbon representation of an X-ray crystallography structure of BRD3-BD2 (PDB ID: 5A7C43). Amino acids important for the crosslinking to Bpa-P1 and Bpa-P2 are shown in magenta and amino acids not required for crosslinking in green. c Crosslinking between wild-type or variant BRD3-BD2 (2 µM) and cyclic peptides (20 µM) after exposure to UV irradiation for 30 min. Samples were separated on an SDS-PAGE gel and visualised by Coomassie staining. d Quantification of (c) representing the mean and standard deviation of three different repeats. e Binding affinities for peptide 3.2B (cyclic-WSWLC(S-)KKYNLIH, K = acetylated lysine) binding to BRD3-BD2 cross-linked to Bpa-P1 or Bpa-P2 measured by SPR. Data represents mean ± standard deviation of the mean.
