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Phosphorylation-Responsive System for Enzyme Activity Detection Based on Peptide-Cucurbit[8]uril Interactions

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The product of the condensation reaction between glycoluril and formaldehyde was first reported in a thesis by Eberhard Meyer in 1904 and then published by Behrend and co-workers in 1905; however, it was not until 1981 that a product was successfully crystallized from the reaction: a macrocycle consisting of 6 glycoluril units bound together by 12 methylene bridges. The macrocycle was named cucurbituril, given its resemblance to a pumpkin (which belongs to the cucurbitaceae family), yet often ...
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Chemical sensing of amino acids, peptides, and proteins provides fruitful information to understand their biological functions, as well as to develop the medical and technological applications. To detect amino acids, peptides, and proteins in vitro and in vivo, vast kinds of chemical sensors including small synthetic binders/sensors, genetically-encoded fluorescent proteins and protein-based semisynthetic biosensors have been intensely investigated. This review deals with concepts, strategies, and applications of protein recognition and sensing using small synthetic binders/sensors, which are now actively studied but still in the early stage of investigation. The recognition strategies for peptides and proteins can be divided into three categories: (i) recognition of protein substructures, (ii) protein surface recognition, and (iii) protein sensing through protein-ligand interaction. Here, we overview representative examples of protein recognition and sensing, and discuss biological or diagnostic applications such as potent inhibitors/modulators of protein-protein interactions.
Article
This paper describes the molecular recognition of the tripeptide Tyr-Leu-Ala by the synthetic receptor cucurbit[8]uril (Q8) in aqueous buffer, with nanomolar affinity, and with exceptional specificity. This combination of characteristics, which also applies to antibodies, is desirable for applications in biochemistry and biotechnology but has eluded supramolecular chemists for decades. Building on prior knowledge that Q8 binds to peptides with N-terminal aromatic residues, a library screen of 105 peptides was designed to test the effects of residues adjacent to N-terminal Trp, Phe, or Tyr. The screen used tetramethylbenzobis(imidazolium) (MBBI) as a fluorescent indicator and resulted in the unexpected discovery that MBBI can serve not only as a turn-off sensor via the simultaneous inclusion of a Trp residue, but also as a turn-on sensor via the competitive displacement of MBBI upon binding of Phe- or Tyr-terminated peptides. The unusual fluorescence response of the Tyr series prompted further investigation by 1H NMR spectroscopy, electrospray ionization mass spectrometry, and isothermal titration calorimetry. From these studies, a novel binding motif was discovered in which only one equivalent of peptide binds to Q8, and the sidechains of both the N-terminal Tyr residue and its immediate neighbor bind within the Q8 cavity. For the peptide Tyr-Leu-Ala, the equilibrium dissociation constant value is 7.2 nM, whereas that of its sequence isomer Tyr-Ala-Leu is 34 μM. The high stability, recyclability, and low cost of Q8 combined with the straightforward incorporation of Tyr-Leu-Ala into recombinant proteins should make this system attractive for the development of biological applications.
Article
Protein-protein interactions (PPIs) are operative at all levels of cellular organization and function. Peptide stapling and hydrogen-bond surrogates, among other approaches, were used to improve the metabolic stability and cell membrane permeability of peptides, which is an important step toward PPI targeting therapeutics. Oligomeric structures such as foldamers represent highly promising, metabolically stable secondary structure mimics, while the structural diversity and biological relevance of natural products will continue to be a rich source for PPI drug discovery. Virtual screening can offer a cost-effective alternative to high-throughput screening (HTS), while supramolecular approaches represent novel orthogonal entries for PPI modulation. Accordingly, depending on the general principle of these screening methods and the biophysical characteristics that the proteins of interest have to possess, a differentiation can be made between surface based and proximity based assays.
Article
We conceptualize a novel approach towards enzyme assays based on the reversible and competitive binding of a fluorescent dye and the substrate as well as product of an enzymatic reaction to a macrocyclic host. This method was termed "supramolecular tandem assay", and has been applied to inhibitor and activator screening, sensor array development, and enantiomeric excess determination of amino acids. The simple and rapid read-out by fluorescence allows their straightforward implementation into high-throughput screening.
Article
This article describes the selective recognition and noncovalent dimerization of N-terminal aromatic peptides in aqueous solution by the synthetic host compound, cucurbit[8]uril (Q8). Q8 is known to bind two aromatic guests simultaneously and, in the presence of methyl viologen, to recognize N-terminal tryptophan over internal and C-terminal sequence isomers. Here, the binding of Q8 to aromatic peptides in the absence of methyl viologen was studied by isothermal titration calorimetry (ITC), (1)H NMR spectroscopy, and X-ray crystallography. The peptides studied were of sequence X-Gly-Gly, Gly-X-Gly, and Gly-Gly-X (X = Trp, Phe, Tyr, and His). Q8 selectively binds and dimerizes Trp-Gly-Gly (1) and Phe-Gly-Gly (4) with high affinity (ternary K = 10(9)-10(11) M(-)(2)); binding constants for the other 10 peptides were too small to be measured by ITC. Both peptides bound in a stepwise manner, and peptide 4 bound with positive cooperativity. Crystal structures of Q8.1 and Q8.4(2) reveal the basis for selective recognition as simultaneous inclusion of the hydrophobic aromatic side chain into the cavity of Q8 and chelation of the proximal N-terminal ammonium group by carbonyl groups of Q8. The peptide sequence selectivity and positively cooperative dimerization reported here are, to the best of our knowledge, unprecedented for synthetic hosts in aqueous solution. Specific peptide recognition and dimerization by synthetic hosts such as Q8 should be important in the study of dimer-mediated biochemical processes and for the separation of peptides and proteins.