No abstract

Inteins are self-processing protein domains that excise themselves out of a precursor polypeptide chain in a multistep pathway termed protein splicing. In the course of this reaction, the sequences flanking the intein, termed N-and Cterminal exteins, are linked with a peptide bond (Scheme 1 A). Inteins perform only a single turn-over, however, they employ catalytic strategies similar to those of enzymes. While inteins have found widespread use in many applications in biotechnology and protein chemistry, important aspects of the mechanism of protein splicing are still not understood. [1] In particular, the N-S (or N-O) acyl shift of the upstream scissile peptide bond into a thioester (or oxoester) remains intriguing (Scheme 1 B). This rearrangement represents the first step of protein splicing in standard inteins [2] and is widely exploited for the generation of protein thioesters. [3] Although thermodynamically unfavored, it does not require any cofactors or energy sources. Several catalytic mechanisms have been proposed for this reaction, including destabilization of the ground state of the scissile peptide bond, general acid-base catalysis to increase the nucleophilicity of the cysteine (or serine) side chain at position 1 of the intein, and stabilization of the tetrahedral intermediate by means of an oxyanion hole (Scheme 1 B). Some of these mechanisms, if not all, can likely be used in combination and may contribute to catalysis to different degrees in different inteins. Here, we pursued a novel chemical approach to directly probe the importance of ground-state destabilization by introducing an alkyl substituent at the amide nitrogen of the scissile peptide bond (Scheme 1 B); a strategy inspired by the recent development of N-S switch devices for the chemical synthesis of peptide thioesters. [4] This chemical manipulation indeed supported the N-S acyl shift, even to the extent that it could complement an otherwise essential part of the catalytic framework of the intein, the highly conserved block B histidine. Together, our findings reveal the role of the histidine in a ground-state destabilization mechanism and rule out other roles previously proposed.The ground-state destabilization of the upstream scissile peptide bond has been proposed on the basis of structural analyses, but its direct investigation remains challenging. In two intein structures, deviations of this peptide bond from the common trans conformation to the energetically less favored cis conformation [5] or a distorted conformation [6] were observed (Scheme 1 B). A solution NMR study by Muir and co-workers on an Mxe GyrA intein precursor that is active in the N-S acyl shift suggested a significant distortion of the peptide bond. [7] Importantly, the highly conserved histidine residue in the block B signature motif (His 75 im Mxe GyrA Intein) was essential for this effect, as a His75Ala mutant was inactive in thioester formation and did not induce any measurable conformational strain on the peptide bond. [7] Block B is one of the conserved seq...

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.

No abstract

Protein trans-splicing using split inteins is a powerful and convenient reaction to chemically modify recombinantly expressed proteins under mild conditions. In particular, semisynthetic protein trans-splicing with one intein fragment short enough to be accessible by solid-phase peptide synthesis can be used to transfer a short peptide segment with the desired synthetic moiety to the protein of interest. In this chapter, we provide detailed protocols for two such split intein systems. The M86 mutant of the Ssp DnaB intein and the MX1 mutant of the AceL-TerL intein are two highly engineered split inteins with very short N-terminal intein fragments of only 11 and 25 amino acids, respectively, and allow the efficient N-terminal labeling of proteins.