Opinion
Cysteine mutants as chemical sensors for ligand–receptor interactions

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Abstract

The incorporation of cysteine residues into membrane receptors by mutagenesis has enabled the development of engineered proteins. Chemical modification of the mutant receptor using a wide range of biochemical and biophysical probes has facilitated functional studies of ligand–receptor interactions. In particular, the substituted-cysteine accessibility method (SCAM) represents a successful example of how to probe transmembrane receptor domains after chemical modification of the mutants with sulfydryl-reacting molecules. We propose an extension of this methodology using site-specific affinity probes that react with cysteine mutants to gain reliable structural information on the binding of a ligand in its receptor site.

Section snippets

Cysteine mutants as chemical sensors for affinity probes

The principle of the approach combining cysteine-scanning mutagenesis and affinity labelling is straightforward: chemical reactivities that are necessary to generate an irreversible reaction between the protein and an affinity marker are created within a putative target binding site. Catalytic enzyme cysteines 1 or naturally occurring cysteine residues in functional receptor sites have been targeted by affinity labels 2, 3; in their absence, such coupling reactions require the incorporation of

Expected outcomes

The expected outcomes from applying this methodology are summarized below. (1) The covalent reactions identify, in a non-ambiguous manner, the amino acids that are in direct contact with the ligand. (2) The method permits topographical analyses of a binding site with precise orientation of the ligand (Fig. 1b). Indeed, recent results of photoaffinity labelling of the agonist binding site on the native nicotinic acetylcholine receptor enabled the determination of the orientation of the

Cysteine scanning and chemical modification: from accessibility towards selectivity

Cysteine scanning has been used to allow the incorporation of chemical probes into membrane receptors 13. To investigate the structure of receptor channels, Karlin and Akabas have developed a method using cysteine-scanning of putative receptor channel elements that were reacted with permeant sulfydryl-reacting molecules to characterize the amino acids oriented towards the lumen of ion channels 14, 15. This method, termed substituted-cysteine accessibility method (SCAM), has been used to

Kinetic considerations of the coupling reaction

The covalent coupling reactions represent typical affinity labelling reactions occurring after reversible ligand–receptor complex formation. Thus, a concentration- and time-dependence of the irreversible reaction should be observed. Such experiments enable the determination of the apparent dissociation constant of the reactive ligand for the mutated receptor and of the apparent coupling rate constant. Similarly, competition experiments should decrease the observed rate of receptor alkylation

Concluding remarks

Site-directed mutagenesis usually targets a single amino acid, selected from sequence databank analyses, giving rise to dramatic functional effects. The corresponding cysteine mutant might also affect receptor functionality, raising the question of the pertinence of using such mutants. To circumvent this problem, we propose to carry out cysteine scanning, mutating the neighbouring residues (i.e. positions ±1–3). Each cysteine mutant is then submitted to controls and assays: (1) the

Glossary

L701324
4-hydroxy-7-chloro-3-(3-phenoxyphenol)-quinolin-2-(1H)-one

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