title: Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.
abstract: Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments.
authors.affiliation: Institut für Chemie, Humboldt-Universität zu Berlin , Brook-Taylor-Strasse 2, D-12489 Berlin, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Reinhardt
authors.affiliation: Institut für Biochemie, Universität Leipzig , Brüderstrasse 34, D-04103 Leipzig, Germany.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Lotze
authors.affiliation: Institut für Biochemie, Universität Leipzig , Brüderstrasse 34, D-04103 Leipzig, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Mörl
authors.affiliation: Institut für Biochemie, Universität Leipzig , Brüderstrasse 34, D-04103 Leipzig, Germany.
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
authors.affiliation: Institut für Chemie, Humboldt-Universität zu Berlin , Brook-Taylor-Strasse 2, D-12489 Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Seitz
mesh_terms: D003545:Cysteine / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D011719:Pyrazines / Q000737:chemistry; D018013:Receptors, Neuropeptide / Q000737:chemistry / Q000378:metabolism; D017476:Receptors, Neuropeptide Y / Q000737:chemistry / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D060327:Solid-Phase Synthesis Techniques
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010455:Peptides; D011719:Pyrazines; D018013:Receptors, Neuropeptide; D017476:Receptors, Neuropeptide Y; D012235:Rhodamines; C083929:neuropeptide FF receptor; C097212:neuropeptide Y-Y1 receptor; C000198:2-amino-3-benzyl-5-(p-hydroxyphenyl)pyrazine; C109491:neuropeptide Y4 receptor; D003545:Cysteine