Photoswitching of Cell Penetration of Amphipathic Peptides by Control of α‑Helical Conformation

We stapled an amphipathic peptide mainly consisting of leucine (L) and lysine (K) by an azobenzene (Ab) linker for photocontrol of the secondary structure. The <i>cis</i>–<i>trans</i> isomerization of the Ab moieties could stabilize and destabilize the α-helical conformation of the LK peptide along with dramatic change of associated peptide structures in a reversible manner by UV–vis irradiation. The cell-penetrating activities of the LK peptide can be readily regulated by the photocontrol, as the stabilized <i>cis</i>-Ab-LK peptide showed remarkable increase of cell penetration compared to the destabilized <i>trans</i>-Ab-LK peptide. The photoswitchable cell-penetrating peptides would provide important structural information for cell permeability as well as an effective targeting strategy for peptide-based pharmaceuticals with spatiotemporal specificity

Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries

Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries

Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries

Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries

Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries

Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries

Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries

Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries