A fluorophore ligase for site-specific protein labeling inside living cells

Abstract

Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structureguided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.fluorescence microscopy | biotechnology | enzyme engineering T echniques for posttranslational labeling of proteins in living cells address some of the shortcomings of green fluorescent protein (GFP) by expanding the repertoire of chemical probes available for protein visualization (1). However, most of these methods, such as HaloTag (2), SNAP/CLIP (3), and DHFR (4), still use large protein tags that sterically interfere with protein trafficking and function, as GFP is known to do (5, 6). FlAsH (7) is the only peptide-based posttranslational labeling method that currently works inside living cells. At 12 amino acids, the FlAsH tag is much smaller than GFP, but poor labeling specificity (7-9), cellular toxicity, and undesired palmitoylation (7) and oxidation (8) of the Cys 4 recognition motif limit its utility.Here we introduce a method for protein labeling that utilizes a peptide tag while preserving high sequence specificity inside living cells. Our method, called PRIME (PRobe Incorporation Mediated by Enzymes), is based on a "fluorophore ligase" that is engineered from the Escherichia coli enzyme lipoic acid ligase (LplA). LplA's natural function is to ligate lipoic acid onto three E. coli proteins involved in oxidative metabolism (10). We previously used LplA for labeling of cell-surface proteins by demonstrating that the wild-type enzyme can ligate an azidoalkanoic acid instead of lipoic acid (11) (Fig. 1A, Middle). Ligated azide could then be chemoselectively derivatized using cyclooctynefluorophore conjugates.In this work, we wished to extend LplA-mediated labeling to intracellular proteins but recognized the challenges associated with our two-step labeling scheme. First, labeling sensitivity is limited by the kinetics of strain-promoted ½3 þ 2 cycloaddition, which has a rate constant of 4.3 × 10 −3 M −1 sec −1 (12). Second, for intracellular applications, two washout steps would be needed: first to remove excess azide, and second to remove excess fluorophore. We s...

Chemical tags: Applications in live cell fluorescence imaging

“…By mutational analysis, the residue W37 was identified to be the most important for expanding the substrate specificity. Based on this finding Uttamapinant et al recently evolved LplA mutants that accept a fluorescent coumarin derivative as substrate [53]. The two most promising candidates, LplA(W37V) and LplA(W37I), have succeeded in intracellular labeling of peptide tagged proteins and did not exhibit cross-reactivity to endogenous sequences or substrates.…”

Genetisch kodierte kupferfreie Klick‐Chemie

“…Das so eingebaute Cyclooctin fungierte dann in einem zweiten Schritt als spezifische Position zur Markierung in Zellen. [12] Die direkte genetische Kodierung von fluoreszierenden nichtnatürlichen Aminosäuren (UASs) hat viele Einschrän-kungen und Nachteile vorheriger Ansätze überwunden, indem sie ausgezeichnete Spezifität, freie Wahl der Position der Modifikation innerhalb des Proteins und -wenn über-haupt -nur minimale strukturelle Beeinträchtigung mit sich bringt. Dies wurde zuerst von Summerer et al durch die Weiterentwicklung eines Leucyl-tRNA/RS-Paars (RS = Synthetase) von E. coli umgesetzt, das die UAS Dansylalanin genetisch in Saccharomyces cerevisiae kodiert.…”

A general strategy for the evolution of bond-forming enzymes using yeast display

“…D espite the many attractive features of protein enzymes as catalysts for organic synthesis (1), as research tools (2)(3)(4), and as an important class of human therapeutics (5,6), the extent and diversity of their applications remain limited by the difficulty of finding in nature or creating in the laboratory highly active proteins that catalyze chemical reactions of interest. A significant fraction of protein catalysts currently used for research and industrial applications was obtained through the directed evolution of natural enzymes (7).…”