Mutational Analysis of Protein Splicing, Cleavage, and Self-Association Reactions Mediated by the Naturally Split Ssp DnaE Intein

Abstract

The ability to separately purify the naturally split Synechocystis sp. PCC6803 (Ssp) DnaE intein domains has allowed detailed examination of both universal and Ssp DnaE intein-specific steps in the protein splicing pathway. By engineering substitutions at both the +1 and penultimate intein positions, we have further characterized intein reaction kinetics in this system. Replacement of the crucial +1Cys with serine decreased N-terminal cleavage and trans-splicing rates; however, this substitution did not prevent splicing or the ability of ZnCl2 to inhibit it. Substitution of the penultimate intein residue (alanine) with a typically conserved histidine did not increase the rate or extent of trans-splicing or cleavage under typical assay conditions. Despite the observation that this histidine aids in asparagine cyclization for other inteins, it did not encourage C-terminal cleavage for the Ssp DnaE intein or uncouple it from N-terminal cleavage. Both the +1Ser and Ala to His mutants were insensitive to ZnCl2 during trans-cleavage experiments, uncoupling a previously linked inhibition in asparagine cyclization from an inhibition in trans-thioesterification detected for the wild-type intein.

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

“…We generated a C1A mutant for each of the four novel split inteins to test whether these proteins could be used for C-terminal cleavage applications. Previously reported naturally split inteins display very slow or no self-cleavage activity at all when mutated at the same position (26,40). Ssp DnaE is the only reported natural split intein rendering a C-terminal cleavage rate of 1.9 ϫ 10 Ϫ4 s Ϫ1 , but only after N-terminal cleavage has occurred.…”

Kinetic Analysis of the Individual Steps of Protein Splicing for the Pyrococcus abyssi PolII Intein

“…This use of temperatureinducible splicing of inteins isolated from extreme thermophiles was also useful in the discovery of the chemical mechanism of splicing (6, 10 -13). Previously, Evans and coworkers (9,14,15) have described the kinetics of the trans-splicing of the intein that interrupts the DnaE protein of Synechocystis sp. PCC6803 (Ssp).…”

Use of protein trans‐splicing to produce active and segmentally 2H, 15N labeled mannuronan C5‐epimerase AlgE4

“…Cleavage of the N-terminal intein part has been described before, [34][35][36] the instability of Int C -R was probably caused by traces of proteolytic enzymes, as the degradation of Int C -R could be prevented by adding protease inhibitors. While none of the thiol-based reducing agents yielded a satisfying level of ligation, the Tris(2-carboxyethyl)phosphine (TCEP) reducing agent gave high yields of trans-splicing without cleavage [ Fig.…”