Side‐chain cyclization of Gln or Asn residues coupled to peptide bond cleavage in an intein and a model peptide

Abstract

Protein splicing is a post‐translational event by which an intervening polypeptide, or intein, facilitates its own excision from the flanking polypeptides, or exteins, and the ligation of the exteins. An epimerase found in Trichodesmium erythraeum contains two inteins, Ndse1 and Ndse2. The first intein, Ndse1, can facilitate protein splicing efficiently in E. coli, even with a native C‐terminal Gln replacing the highly conserved Asn. In addition, activity of the intein may be influenced two downstream Cys residues and the presence of disulfide bonds, as we observed differential reactivity in inteins expressed under reducing and non‐reducing conditions. Substitution of Gln for Asn results in an increase in the rate of C‐terminal bond cleavage for the PolII intein from Pyrococcus abyssi. To study side chain cyclization in a model system, we obtained three pentapeptides of Abz‐His‐Xxx‐ Src‐Lys, where Xxx is Gln, Asn or Ala. Dnp, dinitrophenyl, is attached to the C‐terminal Lys and ortho‐aminobenzoic acid (Abz) serves as the N‐terminal residue. Peptide bond cleavage separates the Abz fluorophore from the Dnp quencher.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KM), and a Holy Cross Alumni/Parents Summer Research Scholarship (LU).