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Cucurbit[7,8]urils are known to form inclusion complexes with hydrophobic amino acids such as Trp, Tyr, Phe, and Met, as well as peptides containing these residues at the N-terminus. Despite their widespread use in protein purification, the affinity of histidine (His) for cucurbit[7,8]urils has not been extensively explored. In this study, X-ray diffraction experiments were conducted to investigate the binding of two histidine moieties to the cucurbit[7]uril (CB7) cavity, resulting in a network of π-π and hydrogen bonds. This assembly was found to induce a His pKa shift of ΔpKa=-4. Histidine weakly bound to CB7 or CB8; however, isothermal titration calorimetry revealed micromolar equilibrium dissociation constant values for CB7 and CB8 when bound to dipeptides containing His at the C-terminus. Conversely, dipeptides with His at the N-terminus exhibited millimolar values. Additionally, the His-Gly-Gly tripeptide formed a 2 : 1 complex with CB7. These findings suggest the potential use of histidine and histidine-containing tags in conjunction with CB7 for various biological applications., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The presence of H atoms connected to either or both of the two N atoms of the imidazole moiety in a histidine residue affects the geometry of the five-membered ring. Analysis of the imidazole moieties found in histidine residues of atomic resolution protein crystal structures in the Protein Data Bank (PDB), and in small-molecule structures retrieved from the Cambridge Structural Database (CSD), identified characteristic patterns of bond lengths and angles related to the protonation state of the imidazole moiety. Using discriminant analysis, two functions could be defined, corresponding to linear combinations of the four most sensitive stereochemical parameters, two bond lengths (ND1-CE1 and CE1-NE2) and two endocyclic angles (-ND1- and -NE2-), that uniquely identify the protonation states of all imidazole moieties in the CSD and can be used to predict which N atom(s) of the histidine side chains in protein structures are protonated. Updated geometrical restraint target values are proposed for differently protonated histidine side chains for use in macromolecular refinement.