Proc. Natl. Acad. Sci. USA 114, 8538–8543 (2017)

Credit: NATIONAL ACADEMY OF SCIENCES

Cyanobacterial DnaE split inteins catalyze the linkage of two polypeptide chains in ultrafast protein trans-splicing reactions, an activity that is exploited in numerous protein engineering applications. However, sequence constraints near the splice site in the second polypeptide chain (C-extein) are still a limiting factor, because large hydrophobic residues at C-extein position +2 are necessary to stabilize the side chain of the catalytically important histidine. Stevens et al., rationalizing that altering side chain dynamics might help to overcome these limitations, have mutated the three loop residues preceding the histidine in the Nostoc punctiforme DnaE split intein Npu and analyzed the mutants in an Escherichia coli selection assay. The NpuGEP loop mutant shows increased splicing rates for unfavorable C-extein +2 residues, and molecular dynamics simulations suggest that the conformational dynamics of the histidine side chain is indeed restricted in NpuGEP. The authors have also introduced the GEP-loop mutation into their previously engineered stable consensus DnaE intein Cfa. The CfaGEP enzyme displays improved protein cyclization activity and facilitates higher yields for in nucleo histone semisynthesis. This mechanism-guided mutagenesis approach may also be applied to other split inteins, thereby potentially expanding the applicability of these important and versatile protein engineering tools.