SPP1623/1: Selective bioconjugation of proteins in live cells via peptide-directed acyl and alkyl transfer reactions

Fluorescently labelled 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 labelling 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 labelling 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 recombinantly expressed proteins (adiponectin), membrane proteins with extracellular (e.g. neuropeptide Y2 receptor) or intracellular (e.g. adiponectin receptor 1) tags. Biotinylation will facilitate the purification. Rapid fluorescence labelling at the N-terminus of membrane proteins will enable studies of protein trafficking by pulse-chase experiments.