Fig. 2: Successful photoconjugation of PpL to an antibody light chain, and successful blocking of an anti-FLAG antibody followed by light activation.

a Different locations were chosen on PpL based on the crystal structure (PDB 1 mhh) to introduce BpA. b A reducing SDS-PAGE gel with 50 µM of each PpL mutant irradiated with 1 µM mouse anti-CD3 antibody shows a photoconjugated product between the light chain and PpL (*) only with the R33BpA location, shown in red. Other tested locations are shown in yellow. All gels are labeled with a mass ladder control in kDa. c Graph showing each amino acid in PpL with the solvent exposure level and number of antibody carbons that are within 1 nm. The higher the solvent exposure and the higher the number of proximal carbons the more likely the amino acid was to be a successful candidate for modification with BpA. Yellow shows which amino acids were modified and red shows amino acid R33 which was ultimately successful with a high surface exposure and number of proximal carbons. The amino acids are also labeled as “Free” or “Bound”, based on a 0.35 nm distance cut-off between the PpL sidechain and antibody atoms. d Reducing SDS-PAGE gel showing 100 µM of PpL-R33BpA (R33) with 4 µM mouse anti-CD3 antibody (Ab) irradiated under 360 nm light for the time indicated. PpL-R33BpA photoconjugated to the light chain is indicated with (*). e The R33BpA mutant with the linker arm discussed above was photoconjugated to the anti-FLAG antibody and then attached via sortase to a photocleavable blocking peptide. Light exposure for 10 min lead to photocleavage and loss of the blocking peptide. f The anti-FLAG antibody alone or modified with the photocleavable blocking peptide (as in e), was treated with 365 nm light for the indicated time, then diluted to 10 nM and analyzed via ELISA for binding (n = 3). The tethered blocking peptide successfully reduced the binding efficiency of the anti-FLAG antibody to its target and was removed with brief light exposure, leading to activation of antibody binding. **P < 0.01 and *P < 0.05 by Welch’s t-test (two-sided). Bar height shows data average. Error bars are standard deviation.