Figure 6: Photodeprotection and sumoylation of folded histone H2B.
From: Aromatic thiol-mediated cleavage of N–O bonds enables chemical ubiquitylation of folded proteins
(a) Scheme depicting photolytic cleavage of the auxiliary protecting group from H2B(A117C)photoaux (5) to generate H2B(A117C)aux (6). (b) ESI-MS spectrum of 5. Calculated for 5, 14,059.2 Da. Observed for 5, 14,062.7±2.8 Da. (c) Circular dichroism spectrum of 5 in 50 mM Na2HPO4, pH 7.5. (d) ESI-MS spectrum of 6. Calculated for 6, 13,924.1 Da. Observed for 6, 13,925.8±2.6 Da. (e) Circular dichroism spectrum of 6 in 50 mM Na2HPO4, pH 7.5. (f) Scheme depicting ligation of 6 to SUMO-3(2–91)C47S-α-thioester to yield H2B(A117C)Su(C47S)aux (7) and subsequent MPAA-mediated auxiliary removal to yield H2B(A117C)Su(C47S) (8) under folded conditions. (g) Coomassie-stained 15% SDS–polyacrylamide gel electrophoresis gel of ligation between H2B(A117C)aux (6) and SUMO3(2–91)C47S-α-thioester under non-denaturing conditions. Lane 1=SUMO-3(2–91)C47S-MES, 2=H2B(A117C)aux, 3=24 h ligation, 4=48 h ligation, 5=24 h MPAA incubation. (h) ESI-MS spectrum of the ligation product, H2B(A117C)Su(C47S)aux (7). Calculated for 7, 24,225.6 Da. Observed for 7, 24,230.9±4.0 Da. (i) ESI-MS spectrum of the final product, H2B(A117C)Su(C47S) (8). Calculated for 8, 24,149.6 Da. Observed for 8, 24,153.1±3.2 Da.
