Recent Trends in Photoaffinity Labeling

Abstract

Investigation of receptor-ligand interactions remains an inexhaustible challenge for chemists and biologists. Structural exploration of biological receptors is the starting point for a better understanding of how they function. Photoaffinity labeling is a biochemical approach to identify and characterize receptors targeting further structural investigations. The primary structure of a receptor protein was typically obtained by reverse genetics after exhaustive purification and sequencing of the N-terminal peptide, which allowed the design of the corresponding oligonucleotide probes. Synthesis of these oligonucleotide probes then led to identification of cDNA clones by hybridization. Following this strategy, several membrane neurotransmitter receptors and constituent polypeptides, present in very small quantities in the central nervous system, were identified and their sequence deduced from the cDNA sequence. Since photoaffinity labeling implies the formation of a covalent bond between a radiolabeled ligand analogue and a receptor binding site, it becomes theoretically possible to isolate and sequence radiolabeled peptides and then synthesize the corresponding oligonucleotide probes. Photoaffinity labeling might avoid the critical solubilization and purification steps of the classical approach. To our knowledge, no such example of primary structure determination based on photoaffinity labeling experiments has been reported. However, the extraordinary developments in gene cloning technologies, in particular homology cloning and expression cloning, have made this approach obsolete and raised the question of new perspectives for photoaffinity labeling technology. In this article we present an update on selected original developments, as well as new challenges for this method. Photoaffinity labeling not only gives access to structural elements but is also a potential tool for the investigation of functional aspects of biological receptors, for example their role in signal transduction mechanisms.

Identification of a Putative Sordarin Binding Site inCandida albicans Elongation Factor 2 by Photoaffinity Labeling

“…Fitting the experimental points to a single hyperbola yielded K d ϭ 1.26 M. This indicates that sordarin binds to EF-2 in a specific manner to a defined binding site within the EF-2 molecule and hence supports performing photoaffinity labeling with EF-2 alone. This affinity, similar to that described for rat liver EF-2 and its natural substrate GTP (K d ϭ 3.0 M) (24), seems to be high enough to reduce the risk of nonspecific labeling (25,26). On the other hand, the presence of ribosomes unveils two classes of binding sites with higher affinity, as deduced from the Scatchard plot (Fig.…”

Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to γ-Aminobutyric Acid Type A Receptors

“…Although photoaffinity labeling is a powerful tool to identify amino acids contributing directly to drug binding sites in proteins, results can be biased because the photoreactive intermediate may react preferentially with only certain amino acid side chains, and there is also the possibility that highly reactive side chains distant from the binding site may be labeled (10,11).…”

“…Photoaffinity labeling, which allows the identification of residues in proximity to drug binding sites (10,11), has been used to identify two GABA A R amino acids covalently modified by the etomidate analog [ 3 H]azietomidate (12): ␣1Met-236 within ␣M1 and ␤Met-286 within ␤M3. Photolabeling of these residues was inhibited equally by nonradioactive etomidate and enhanced proportionately by GABA present in the assay, consistent with the presence of these two residues in a common drug binding pocket that would be located at the interface between the ␤ and ␣ subunits in the transmembrane domain (12).…”

[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

“…Photoaffinity labeling provides an alternative means to identify amino acids contributing to a drug binding site (18,19) and has been used to determine the orientation of drugs bound in the ABS of Torpedo nAChR (20). Epibatidine binds with very high affinity (ϳ10 pM) to heteromeric neuronal nAChRs (e.g.…”