Steric bulk can compensate for a catalytically critical histidine in an intein's active site and promote the N–S acyl shift.

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split-intein-mediated SICLOPPS technology provides a generic access to fully genetically encoded head-to-tail cyclized peptides and large libraries thereof (SICLOPPS=split-intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light-dependent intein using the photocaged tyrosine derivative ortho-nitrobenzyltyrosine incorporated at an internal, non-catalytic position is now reported. Stable intein precursors were purified from the E.coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.

Protein splicing performed by inteins provides powerful opportunities to manipulate protein structure and function, however, detailed mechanistic knowledge of the multistep pathway to help engineering optimized inteins remains scarce. A typical intein has to coordinate three steps to maximize the product yield of ligated exteins. We have revealed a new type of coordination in the Ssp DnaB intein, in which the initial N- S acyl shift appears rate-limiting and acts as an up-regulation switch to dramatically accelerate the last step of succinimide formation, which is thus coupled to the first step. The structure-activity relationship at the N-terminal scissile bond was studied with atomic precision using a semisynthetic split intein. We show that the removal of the extein acyl group from the α-amino moiety of the intein's first residue is strictly required and sufficient for the up-regulation switch. Even an acetyl group as the smallest possible extein moiety completely blocked the switch. Furthermore, we investigated the M86 intein, a mutant with faster splicing kinetics previously obtained by laboratory evolution of the Ssp DnaB intein, and the individual impact of its eight mutations. The succinimide formation was decoupled from the first step in the M86 intein, but the acquired H143R mutation acts as a brake to prevent premature C-terminal cleavage and thereby maximizes splicing yields. Together, these results revealed a high degree of plasticity in the kinetic coordination of the splicing pathway. Furthermore, our study led to the rational design of improved M86 mutants with the highest yielding trans-splicing and fastest trans-cleavage activities.

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split-intein-mediated SICLOPPS technology provides a generic access to fully genetically encoded head-totail cyclized peptides and large libraries thereof (SICLOPPS = split-intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, lightdependent intein using the photocaged tyrosine derivative ortho-nitrobenzyltyrosine incorporated at an internal, noncatalytic position is now reported. Stable intein precursors were purified from the E. coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.

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