A rapid, site-selective and efficient route to the dual modification of DARPins.

Designed ankyrin repeat proteins (DARPins) are valuable tools in both biochemistry and medicine. Herein we describe a rapid, simple method for the dual modification of DARPins by introduction of cysteine mutations at specific positions that results in a vast difference in their thiol nucleophilicity, allowing for clean sequential modification.

Reversible protein affinity-labelling using bromomaleimide-based reagents.

Reversible protein biotinylation is readily affected via conjugation with a bromomaleimide-based reagent followed by reductive cleavage. The intermediate biotinylated protein constructs are stable at physiological temperature and pH 8.0. Quantitative reversibility is elegantly delivered under mild conditions of using a stoichiometric amount of a bis-thiol, thus providing an approach that will be of general interest in chemical biology and proteomics.

A novel approach to the site-selective dual labelling of a protein via chemoselective cysteine modification.

Local protein microenvironment is used to control the outcome of reaction between cysteine residues and 2,5-dibromohexanediamide. The differential reactivity is exploited to introduce two orthogonal reactive handles onto the surface of a double cysteine mutant of superfolder green fluorescent protein in a regioselective manner. Subsequent elaboration with commonly used thiol and alkyne containing reagents affects site-selective protein dual labelling.

Bromomaleimides: new reagents for the selective and reversible modification of cysteine.

Bromomaleimides react rapidly and selectively with cysteine to afford thiomaleimides which can be cleaved with a phosphine to regenerate the cysteine or treated with a base to afford dehydroalanine.