Beck-Sickinger/Seitz

Selective bioconjugation of proteins in live cells via peptide-directed acyl and alkyl transfer reactions

Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. The fluorophore is introduced either as autofluorescent protein or by means of labeling reactions targeted against specific tag sequences. Of major concern is the size of the fluorophore-tag. The tag should be small to prevent interference with protein function. We will develop a bioorthogonal labeling reaction that allows the introduction of virtually any reporter group, requires only small tag sequences (max 23 aa), occurs with high tag specificity and proceeds within minutes (rather than hours). High reaction rates are required in pulse chase experiments, which allow the analysis of receptor internalization in living cells.

To achieve the goals, we will take advantage of peptide-templated reactions. A cysteine-containing acceptor tag will be fused to the protein of interest. A second, so-called donor peptide binds the acceptor with nanomolar affinity. This interaction triggers the transfer of a thioester- or sulfonate-linked reporter group from the donor peptide to the acceptor peptide. The acceptor peptides will be fused to  membrane proteins with extracellular (e. g. neuropeptide Y2 receptor) or intracellular (e. g. adiponectin receptor 1) tags.Rapid fluorescence labeling with dirfferent fluorophores will facilitate protein localization within living cells and enable studies of protein trafficking by pulse-chase experiments. Transfer of PNA moieties will extend the application to allow e.g. quantification of proteins at cell surfaces or to get insights into dimerization of membrane proteins. Furthermore biotinylation will facilitate the purification.

Prof. Dr. Annette G. Beck-Sickinger
Universität Leipzig

Tel. +49 341 97-36900
Fax +49 341 97-36909

Email Prof. Beck-Sickinger

Prof. Dr. Oliver Seitz
Humboldt Universität zu Berlin

Tel: +49 30 2093 - 7446
Fax +49 30 2093 - 7266

Email Prof. Seitz

Publications within the SPP 1623 project

P. Wolf, A. Mohr, G. Gavins, V. Behr, K. Mörl, O. Seitz, A.G. Beck-Sickinger
ChemBioChem2021, accepted
Orthogonal peptide-templated labeling elucidates lateral ETAR/ETBR proximity and reveals altered downstream signalling
Link to the article

G.C. Gavins, K. Gröger, M. Reimann, M.D. Bartoschek, S. Bultmann, O. Seitz
RSC Chem. Biol.2021,2(4), 1291-1295
Orthogonal coiled coils enable rapid covalent labelilng of two distinct membrane proteins with peptide nucleic acid barcodes
Link to the article

T. F. Fischer, A.S. Czerniak, T. Weiß, C.T. Schoeder, P. Wolf, O. Seitz O, J. Meiler, A.G. Beck-Sickinger
Cell Mol Life Sci. 2021, 78(17-18), 6265-6281
Ligand-binding and -scavenging of the chemerin receptor GPR1
Link to the article

P. Wolf, A.G. Beck-Sickinger
J. Pept Sci.2021, 27(7), e3325
The ring size of monocyclic ET-1 controls selectivity and signaling efficiency at both endothelin receptor subtypes
Link to the article

P. Wolf, G. Gavins, A.G. Beck-Sickinger, O. Seitz
Chembiochem 2021, 22(10), 1717-1732
Strategies for Site-Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags
Link to the article

G.C. Gavins, K. Gröger, M.D. Bartoschek, P. Wolf , A.G. Beck-Sickinger, S. Bultmann, O. Seitz
Nat Chem. 2021, 13(1), 15-23
Live cell PNA labelling enables erasable fluorescence imaging of membrane proteins
Link to the article

J. Lotze, U. Reinhardt, O. Seitz, K. Mörl,A.G. Beck-Sickinger
ACS Chem Biol. 2018, 13(3), 618-627.
Time-Resolved Tracking of separately Internalized Neuropeptide Y(2) Receptors by Two-Color Pulse-Chase.
Link to the article

K. Gröger, G. Gavins, O. Seitz
Angew. Chem. Int. Ed.2017, 56, 14217-14221
Strand Displacement in Coiled-Coil Structures - Controlled Induction and Reversal of Proximity
Link to the article

J. Lotze, U. Reinhardt, O. Seitz, A.G. Beck-Sickinger
Mol Biosyst. 2016, 12(6), 1731-45
Peptide-tags for site-specific protein labelling in vitro and in vivo
Link to the article

U. Reinhardt, J. Lotze, K. Mörl, A.G. Beck-Sickinger, O. Seitz
Bioconjug. Chem. 2015, 26, 2106-2117
Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.
Link to the article

V. te Kamp, H.-G. Jahnke, D. Krinke, K.B. Kostelnik, A.G. Beck-Sickinger, A.A. Roblitzki
Biosens Bioelectron 2014, 14, 662-669
Quantitative impedimetric NPY-receptor activation monitoring and signaling profiling in living cells.
Link to the article

V. Mäde, K. Bellmann-Sickert, A. Kaiser, J. Meiler, A.G. Beck-Sickinger
ChemMedChem 2014, 9, 2463-2474
Position and length of fatty acids strongly affect receptor selectivity pattern of human pancreatic polypeptide analogues.
Link to the article

U. Reinhardt, J. Lotze, S. Zernia, K. Mörl, A.G. Beck-Sickinger, O. Seitz
Angew. Chem. Int. Ed.2014, 53, 10237-10241.
Peptide-templated acyl transfer: a chemical method for the labeling of membrane proteins.
Link to the article

V. Mäde, S. Babilon, N. Jolly, L. Wanka, K. Bellmann-Sickert, L.E. Diaz Gimenez, K. Mörl, H.M. Cox, V.V. Gurevich, A.G. Beck-Sickinger
Angew. Chem. Int. Ed.2014, 53, 10067-10071
Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias.
Link to the article