Platform for Orthogonal <i>N</i>-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents
Alena Istrate, Michael B. Geeson, Claudio D. Navo, Bárbara B. Sousa, Marta C. Marques, Ross Taylor, Toby Journeaux, Sebastian Oehler, Michael R. Mortensen, Michael J. Deery, Andrew D. Bond, Francisco Corzana, Gonzalo Jiménez‐Osés, Gonçalo J. L. Bernardes
Abstract
, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the β-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.