Structure-assisted ligand-binding analysis using fluorogenic photoaffinity labeling.

Photoaffinity labeling (PAL) technique using a fluorogenic cross-linker is used to monitor the nucleotide-binding pocket within a protein. A coumarin fluorophore formed in the binding domain due to ultraviolet (UV) irradiation has been shown to accelerate the sequencing of the labeled peptide as well as identification of the labeled site by liquid chromatography (LC)-tandem mass spectrometry (MS), in addition to providing information on the ligand binding state. Selective monitoring of the predefined fluorescence peaks among the numerous digests obtained from high performance liquid chromatography (HPLC) clearly indicates the binding capability of the ligand to the entire protein as well as to the corresponding binding domain under various conditions. In the current study, ligand-binding analysis confirmed by the structural information of the binding state has been demonstrated using fluorogenic ATP/ADP photoactivatable probes under allosteric regulation of multiple substrates in the enzyme glutamate dehydrogenase (GDH).

Affinity-based fluorogenic labeling of ATP-binding proteins with sequential photoactivatable cross-linkers.

A specific illumination approach has been developed for identification of adenosine triphosphate (ATP)-binding proteins. This strategy utilizes a tandem photoactivatable unit that consists of a diazirine group as a carbene precursor and an o-hydroxycinnamate moiety as a coumarin precursor. The photolysis of diazirine induces a specific cross-link on target proteins and is followed by photoactivation of coumarin generation with a concomitant release of the pre-installed affinity ligand. The ATP, installed with this cross-linker at the γ-position, successfully transferred a coumarin onto ATP-binding proteins using only UV-irradiation.

Rapidly photoactivatable ATP probes for specific labeling of tropomyosin within the actomyosin protein complex.

Tropomyosin-specific photoaffinity adenosine triphosphate (ATP) probes have been first developed, in which a diazirine moiety is incorporated into the γ-phosphate group as a rapidly carbene-generating photophore. These probes clearly labeled tropomyosin in the presence of other actomyosin components, that is, myosin, actin, and troponins. The specific labeling of tropomyosin was easily identified by selective trapping of the photo-incorporated ATP probe on Fe(3+)-immobilized metal ion affinity chromatography (IMAC) beads. The characteristic nature of tropomyosin-specific photocross-linking was further confirmed with a biotin-carrying derivative of the ATP probe. These data suggest that the tropomyosin on the actin filament assembly is located in close proximity to the ATP binding cavity of myosin.

Photochemical construction of coumarin fluorophore on affinity-anchored protein.

A simple and unique construction of a coumarin fluorophore on a prey protein has been achieved by sequential photoreactions using a nonfluorescent bait protein. The bait protein was first modified with a novel diazirine-based photo-cross-linker containing an o-hydroxycinnamoyl unit. The strategy involves two wavelength-dependent photoreactions: photolysis of the diazirine group and E to Z photoisomerization of the cinnamoyl group. The cross-linked interacting partners were cleaved by the consecutive steps of photoisomerization and thermal lactonization accompanied with the creation of a coumarin derivative on the prey protein as a fluorescent tag. Finally, the methodology was successfully applied for coumarin labeling of antilysozymes expressed on living B cells by using photoactivatable lysozymes.