Molecular Recognition of Insulin by a Synthetic Receptor

Abstract

The discovery of molecules that bind tightly and selectively to desired proteins continues to drive innovation at the interface of chemistry and biology. This paper describes the binding of human insulin by the synthetic receptor cucurbit[7]uril (Q7) in vitro. Isothermal titration calorimetry and fluorescence spectroscopy experiments show that Q7 binds to insulin with an equilibrium association constant of 1.5 × 106 M-1 and with 50-100-fold selectivity versus proteins that are much larger but lack an N-terminal aromatic residue, and >1000-fold selectivity versus an insulin variant lacking the N-terminal phenylalanine (Phe) residue. The crystal structure of the Q7•insulin complex shows that binding occurs at the N-terminal Phe residue and that the N-terminus unfolds to enable binding. These findings suggest that site-selective recognition is based on the properties inherent to a protein terminus, including the unique chemical epitope presented by the terminal residue and the greater freedom of the terminus to unfold, like the end of a ball of string, to accommodate binding. Insulin recognition was predicted accurately from studies on short peptides and exemplifies an approach to protein recognition by targeting the terminus.

Nanomolar Binding of Peptides Containing Noncanonical Amino Acids by a Synthetic Receptor

“…Recently our group has shown that this selectivity can translate from peptides to folded proteins via the unraveling of the terminus to accommodate Q7. 6 Despite the excellent selectivity observed in these systems, the lowest Kd values (for N-terminal phenylalanine) are in the 0.1-1 M range. Such affinities by artificial receptors in aqueous solution are considered relatively high by current standards in the field, 7 but a 100-fold or better boost in affinity would significantly increase the viability of in vivo applications.…”

A Binary Bivalent Supramolecular Assembly Platform Based on Cucurbit[8]uril and Dimeric Adapter Protein 14‐3‐3

“…By contrast, no such difference in binding affinity was observed with the N‐terminal acetylated peptide (Ac‐FGG‐ERα) upon the addition of Q8 (Figure 2 C). Furthermore, the analogous cucurbit[7]uril, Q7, which binds only one FGG moiety,30 did not enhance the apparent affinity of FGG‐ERα for the 14‐3‐3 protein (Figure 2 E). Combined, these reference experiments provide experimental evidence that the enhancement of the apparent affinity between the FGG‐ERα peptide cargo and 14‐3‐3 protein by Q8 relies exclusively on its postulated bivalent FGG recognition.…”

Sequence-Specific Inhibition of a Nonspecific Protease

“…[6][7][8] We and others have studied the capacity of Q7 to bind to amino acids, peptides, and proteins and found that Q7 prefers to bind Nterminal phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) residues, by incorporating the sidechain within the nonpolar Q7 cavity and chelating the N-terminal ammonium group with Q7 carbonyl oxygens. [9][10][11][12][13][14] Nau and coworkers have shown that Q7 can slow the activity of an endopeptidase, trypsin, and an exopeptidase, leucine aminopeptidase (LAP), by binding to their respective substrates. 15,16 In both cases, they observed only partial and short-lived inhibition.…”