A Novel Approach for Affinity-Based Screening of Target Specific Ligands:  Application of Photoreactive d-Glyceraldehyde-3-phosphate Dehydrogenase

Abstract

A novel application of the photoaffinity technique has been developed for the efficient discovery of small ligand and macromolecule interaction. The approach, photoaffinity capture, uses a photoreactive protein together with immobilized ligand for the rapid screening of competitive inhibitors. The set of photoreactive glyceraldehyde-3-phosphate dehydrogenase (photo-GAPDH) and immobilized dye ligand was prepared and examined as a model system. The photo-GAPDH was shown to efficiently capture the immobilized ligand. When nonimmobilized competitive ligands were included in the system, the capture was prevented in accordance with the affinity of the ligands. The present approach would provide an efficient tool for affinity-based screening of ligand libraries.

Simple and Versatile Method for Tagging Phenyldiazirine Photophores

“…The tryptophan residue in the original sequence of CaMBP 10 introduced into the peptide via disulfide linkage by substituting the Trp 3 with cysteine. 18,19 Figure 1 shows the photocross-linking of CaM with 9 visualized by chemiluminescence utilizing the interaction of HRP-conjugated avidin and biotin attached to CaMBP after blotting onto a PVDF membrane. Photolysis rapidly proceeded upon UV-A irradiation with 30 W/m 2 of 360 nm light at 0°C and was complete in a few minutes (see Supporting Information).…”

Photochemical Nucleophile Mapping: Identification of Tyr311 Within the Catalytic Domain of Rabbit Muscle Glyceraldehyde‐3‐phosphate Dehydrogenase†

“…For the photochemical nucleophile mapping of rabbit‐muscle GAPDH, fluoronitrophenyl derivatives having a SH‐reactive methanethiosulfonyl group ( 1a and 1b , Fig. 1) were designed for attaching photophores to the catalytic Cys149 via disulfide linkage (12). Because nucleophilic aromatic photosubstitution takes place at the fluorinated position of the benzene ring, ortho‐ and para ‐derivatives were prepared to compare labeling efficiency and ability depending on the distance from Cys149 to nucleophiles.…”

Development and Leading-Edge Application of Innovative Photoaffinity Labeling

“…Photoaffinity labeling is complementary with other approaches of structural biology and could open rational new strategies for drug discovery and development. The development of diazirine family also extends its application to chip technology for inhibitor screening, 72) one-step fabrication of inert polymer surface, 73) and fluorogenic photoaffinity labeling.…”