Developing approaches to the molecular recognition of peptides with high affinity and selectivity is essential in many areas of modern chemical biology. Previously we found that cucurbit[7]uril (Q7) and cucurbit[8]uril (Q8) is able to recognize peptides at the N-terminus with equilibrium dissociation constant (K_d) values in the low micromolar to low nanomolar range. The N-terminus of proteins, however, is encoded for a methionine residue and is commonly post-translationally modified, both of which can complicate the engineering of a minimal affinity tag for cucurbit[n]uril binding. Prior work showed that Q8 can bind nonterminal dipeptide sites of sequence YL and MY with low micromolar affinity. Here we explore the scope and limitations of Q8 binding to nonterminal dipeptide sites. A peptide library containing 64 unique tetrapeptides of sequence GX₁X₂G (X = F, Y, W, M, L, P, K, and R) was synthesized and screened for Q8 binding using a fluorescent indicator displacement (FID) assay. Several complexes showed larger changes in fluorescence response than the best dipeptide site previously characterized, YL, which prompted us to study the system in more depth. To that end, a series of heptapeptides of sequence GGX₁X₂GGY was synthesized and purified, and Q8 binding was characterized by a combination of isothermal titration calorimetry, ¹H NMR and circular dichroism spectroscopy, and mass spectrometry. We found that the peptides GGFKGGY and GGKFGGY Q8 in a 1:1 Q8:peptide stoichiometric ratio, and the peptide is folded to allow both F and K side chains to bury within the cavity of Q8. Importantly, these peptides bind Q8 with K_d values lower than 100 nM, which to the best of our knowledge is the strongest affinity binding of a nonterminal site on a peptide by any synthetic receptor. This finding should facilitate the design and engineering of minimal protein affinity tags. The results of these studies and the current state of the project will be presented.

Sarah Chang

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