title: Two-step protein labeling by using lipoic acid ligase with norbornene substrates and subsequent inverse-electron demand Diels-Alder reaction.
abstract: Inverse-electron-demand Diels-Alder cycloaddition (DAinv ) between strained alkenes and tetrazines is a highly bio-orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two-step labeling protocol for the site-specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplA(W37V) ), followed by the covalent attachment of tetrazine-modified fluorophores to the norbornene moiety through the bio-orthogonal DAinv  . We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13-residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two-step labeling strategy to label proteins in cell lysates in a site-specific manner and performed cell-surface labeling on living cells.
authors.affiliation: Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg (Germany).
authors.forename: Marcel
authors.initials: M
authors.lastname: Best
authors.affiliation: Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg (Germany).
authors.forename: Anna
authors.initials: A
authors.lastname: Degen
authors.affiliation: Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg (Germany).
authors.forename: Mathis
authors.initials: M
authors.lastname: Baalmann
authors.affiliation: Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg (Germany).
authors.forename: Tobias T
authors.initials: TT
authors.lastname: Schmidt
authors.affiliation: Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg (Germany). wombacher@uni-heidelberg.de.
authors.forename: Richard
authors.initials: R
authors.lastname: Wombacher
mesh_terms: D004579:Electron Transport; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D009636:Norbornanes / Q000737:chemistry* / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*; D013466:Sulfurtransferases / Q000235:genetics / Q000378:metabolism*
chemical_list: D009636:Norbornanes; D011506:Proteins; C046060:2-norbornene; D013466:Sulfurtransferases; C116435:lipoic acid synthase
keywords: fluorescent probes; inverse-electron-demand Diels-Alder cycloaddition; ligases; protein labeling; tetrazines