The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP–substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, the irreversible attachment of the probe to HaloTag precludes imaging applications that require transient binding of the probe and comes with the risk of irreversible photobleaching. Here, we introduce exchangeable ligands for fluorescence labeling of HaloTag (xHTLs) that reversibly bind to HaloTag and that can be coupled to rhodamines of different colors. In stimulated emission depletion (STED) microscopy, probe exchange of xHTLs allows imaging with reduced photobleaching as compared to covalent HaloTag labeling. Transient binding of fluorogenic xHTLs to HaloTag fusion proteins enables points accumulation for imaging in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT and STED microscopy. xHTLs thus open up new possibilities in imaging across microscopy platforms for a widely used labeling approach.

Diels−Alder reactions with inverse electron demand (DA inv ) have emerged as an indispensable tool for bioorthogonal labeling and the manipulation of biomolecules. In this context, reactions between tetrazines and strained dienophiles have received attention because of high reaction rates. Current methods for the DA inv -mediated functionalization of proteins suffer from slow reactivity, impaired stability, isomerization, or elimination of the incorporated strained dienophiles. We report here a versatile platform for the posttranslational, highly selective, and quantitative modification of proteins with stable dienes. New synthetic access to minimal size tetrazine and triazine derivatives enabled us to synthesize tailored diene substrates for the lipoic acid protein ligase A (LplA) from Escherichia coli, which we employ for the rapid, mild, and quantitative bioconjugation of proteins by DA inv . The presented method benefits from the minimal tag size for LplA recognition and can be applied to proteins from any source organism. We demonstrate its broad suitability by site-specific in vitro protein labeling and live cell labeling for fluorescence microscopy. With this work we expand the scope of DA inv bioorthogonal chemistry for site-specific protein labeling, providing additional experimental flexibility for preparing well-defined bioconjugates and addressing biological questions in complex biological environments.

Bioorthogonal chemistry holds great potential to generate difficult‐to‐access protein–protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels–Alder cycloaddition with inverse electron demand (DAinv). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin‐based targeted cancer therapies.

We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum. Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays.

We introduce exchangeable ligands for fluorescence labeling of HaloTag7 as an alternative to covalently bound probes. The exchangeable ligands open up new possibilities in imaging for a widely used labeling approach, including applications in points accumulation for imaging in nanoscale topography (PAINT), MINFLUX and live-cell, multi-frame stimulated emission depletion (STED) microscopy. We furthermore introduce orthogonal pairs of exchangeable ligands and HaloTags for dual-color PAINT and STED microscopy.

Bioorthogonal chemistry holds great potential to generate difficult‐to‐access protein–protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels–Alder cycloaddition with inverse electron demand (DAinv). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin‐based targeted cancer therapies.

A highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels-Alder cycloaddition with inverse electron demand (DA_inv). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines.