title: Depsipeptide substrates for sortase-mediated N-terminal protein ligation.
abstract: Technologies that allow the efficient chemical modification of proteins under mild conditions are widely sought after. Sortase-mediated peptide ligation provides a strategy for modifying the N or C terminus of proteins. This protocol describes the use of depsipeptide substrates (containing an ester linkage) with sortase A (SrtA) to completely modify proteins carrying a single N-terminal glycine residue under mild conditions in 4-6 h. The SrtA-mediated ligation reaction is reversible, so most labeling protocols that use this enzyme require a large excess of both substrate and sortase to produce high yields of ligation product. In contrast, switching to depsipeptide substrates effectively renders the reaction irreversible, allowing complete labeling of proteins with a small excess of substrate and catalytic quantities of sortase. Herein we describe the synthesis of depsipeptide substrates that contain an ester linkage between a threonine and glycolic acid residue and an N-terminal FITC fluorophore appended via a thiourea linkage. The synthesis of the depsipeptide substrate typically takes 2-3 d.
authors.affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Williamson
authors.affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Webb
authors.affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: W Bruce
authors.initials: WB
authors.lastname: Turnbull
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000494:pharmacology*; D047630:Depsipeptides / Q000096:biosynthesis* / Q000737:chemistry*; D005998:Glycine / Q000378:metabolism; D008956:Models, Chemical; D015394:Molecular Structure; D015202:Protein Engineering / Q000379:methods*; D013890:Thiourea
chemical_list: D001426:Bacterial Proteins; D047630:Depsipeptides; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D013890:Thiourea; D005998:Glycine