Protein recognition by cucurbit[6]uril: high affinity N-terminal complexation

The donut-shaped cucurbit[n]urils (Qn,n= 6-8) are rigid macrocyclic receptors with widespread use in protein recognition. To date, most applications have centred on the encapsulation of N-terminal aromatic residues byQ7orQ8. Less attention has been placed onQ6, which can recognize lysine side chains due to its high affinity for alkylamines. In this work, we investigated protein-Q6complexation by using NMR spectroscopy. Attempts to crystallize protein-Q6complexes were thwarted by the crystallization ofQ6. We studied four proteins that vary in size, net charge, and lysine content. In addition toQ6interactions with specific Lys or dimethylated Lys residues, we report striking evidence for N-terminal recognition. High affinity (micromolar) binding occurred with the N-terminal Met-Lys motif present in one of the four model proteins. Engineering this feature into another model protein yielded a similar high affinity site. We also present evidence forQ8binding at this N-terminal feature. These data expand the cucurbituril toolkit for protein sensing.