Fig. 5: Identifying direct interactome of SELM via eFSY induced live cell cross-linking. | Nature Communications

Fig. 5: Identifying direct interactome of SELM via eFSY induced live cell cross-linking.

From: Characterize direct protein interactions with enrichable, cleavable and latent bioreactive unnatural amino acids

Fig. 5

a The design of the SELM probe in which eFSY was incorporated at site 48 and Cys45 was mutated to Ala (‘Sec’ represents selenocysteine). b Western blot analysis of HEK293T cell lysates expressing SELM(C45A-Sec48U). c Strep-tag purified protein complexes cross-linked by the SELM probe were labeled with biotin, followed by western blot analysis. d Inter-protein cross-linked peptides identified by MS. e Validation of PPI between SELM and binding proteins (HSPA5, PRDX4, PLGT3 and CALR). f SELM-PRDX4 cross-linking sites mapped onto the protein structure (PDB ID 3TJF). The cross-linking sites were shown in magenta sticks. Cysteines of PRDX4 were shown in yellow sticks. g Mutants (M1-6) of PRDX4 (picture adapted from PMID: 21057456). h Disulfide trapping experiment showing SELM cross-linked with PRDX4 by inter-protein disulfide bond. Source data are provided as a Source Data file.

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