title: Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide.
abstract: An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore).
authors.forename: Giang K T
authors.initials: GK
authors.lastname: Nguyen
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore).
authors.forename: Yuan
authors.initials: Y
authors.lastname: Cao
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore).
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore).
authors.forename: Chuan Fa
authors.initials: CF
authors.lastname: Liu
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore). jptam@ntu.edu.sg.
authors.forename: James P
authors.initials: JP
authors.lastname: Tam
mesh_terms: D000595:Amino Acid Sequence; D047630:Depsipeptides / Q000737:chemistry*; D008025:Ligases / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
chemical_list: D047630:Depsipeptides; D010455:Peptides; D011506:Proteins; D008025:Ligases
keywords: butelase 1; labelling; ligation; thiodepsipeptide