Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling

Abstract

Weak and transient protein interactions are involved in dynamic biological responses and are an important research subject; however, methods to elucidate such interactions are lacking. Proximity labeling is a promising technique for labeling transient ligand–binding proteins and protein–protein interaction partners of analytes via an irreversible covalent bond. Expanding chemical tools for proximity labeling is required to analyze the interactome. We developed several photocatalytic proximity-labeling reactions mediated by two different mechanisms. We found that numerous dye molecules can function as catalysts for protein labeling. We also identified catalysts that selectively modify tyrosine and histidine residues and evaluated their mechanisms. Model experiments using HaloTag were performed to demonstrate photocatalytic proximity labeling. We found that both ATTO465, which catalyzes labeling by a single electron transfer, and BODIPY, which catalyzes labeling by singlet oxygen, catalyze proximity labeling in cells.

Quantitative Assays for Catalytic Photo-Oxygenation of Alzheimer Disease-Related Tau Proteins

“…Considering that photo-oxygenation of His is more specifically introduced by the catalysts than that of Met, , we envisioned quantifying photo-oxygenated His in tau with a fluorescent molecule. Sato et al reported that a nucleophilic small molecule, 1-methyl-4-arylurazole (MAUra)-derivative 5 , selectively reacts with intermediate 6 generated through the reaction of His with 1 O 2 (Figure a). − The modified sites can be further labeled with the fluorescent molecule, tetramethylrhodamine (TAMRA)-derivative 7 , through azide–alkyne cycloaddition (AAC) to afford 8 (Figure a). − …”

Photocatalytic Structures for Protein Modifications

“…BODIPY also exhibited excellent catalytic efficiency for the modification of histidine residues. [74]…”

“…Among the candidates tested, including Ru(bpy) 3 Cl 2 , fluorescein, dibromofluorescein, rhodamine 123, rhodamine B, BODIPY, halo‐BODIPYs, iodo‐coumarin, eosin Y, Rose Bengal, acriflavine (ATTO465), and riboflavin, acriflavine (ATTO465) was found to be the most efficient for catalyzing tyrosine residues. BODIPY also exhibited excellent catalytic efficiency for the modification of histidine residues [74] …”

Chemical and Biological Strategies for Profiling Protein‐Protein Interactions in Living Cells

“…Photosensitizers can generate reactive oxygen species via a complex process of photoreactions. [94] Subsequently, the active singlet oxygen with short lifetime and limited diffusion radius (t 1/2 <0.6 μs in cells) [95] can promiscuously oxidize the neighboring amino acid residues such as tyrosine [96] and histidine,[ 97 , 98 ] which leads to umpolung polarity and generates the electrophilic moiety that can further react with amino probe nucleophiles, finally achieving the labeling of interacting proteins. [99] To date, several protein‐based photosensitizers, such as the engineered fluorescent proteins KillerRed or TagRFP, the flavin‐binding proteins miniSOG or Pp2FbFP L30M , have attracted wide interests as they are genetically encodable and can be expressed in a fusion protein manner in cells for targeted protein photo‐oxidative activation/desensitization [100] (Figure 3 ).…”