Protein-Calixarene Complexation: From Recognition to Assembly

Abstract

This Account summarizes the progress in protein-calixarene complexation, tracing the developments from binary recognition to the glue activity of calixarenes and beyond to macrocycle-mediated frameworks. During the past 10 years, we have been tackling the question of protein-calixarene complexation in several ways, mainly by cocrystallization and X-ray structure determination as well as by solution state methods, NMR spectroscopy, isothermal titration calorimetry (ITC), and light scattering. Much of this work benefitted from collaboration, highlighted here. Our first breakthrough was the cocrystallization of cationic cytochrome c with sulfonato-calix[4]arene leading to a crystal structure defining three binding sites. Together with NMR studies, a dynamic complexation was deduced in which the calixarene explores the protein surface. Other cationic proteins were similarly amenable to cocrystallization with sulfonato-calix[4]arene, confirming calixarene-arginine/lysine encapsulation and consequent protein assembly. Calixarenes bearing anionic substituents such as sulfonate or phosphonate, but not carboxylate, have proven useful.Studies with larger calix[n]arenes (n = 6, 8) demonstrated the bigger better binder phenomenon with increased affinities and more interesting assemblies, including solution-state oligomerization and porous frameworks. While the calix[4]arene cavity accommodates a single cationic side chain, the larger macrocycles adopt different conformations, molding to the protein surface and accommodating several residues (hydrophobic, polar, and/or charged) in small cavities. In addition to accommodating protein features, the calixarene can bind exogenous components such as polyethylene glycol (PEG), metal ions, buffer, and additives. Ternary cocrystallization of cytochrome c, sulfonato-calix[8]arene, and spermine resulted in altered framework fabrication due to calixarene encapsulation of the tetraamine. Besides host-guest chemistry with exogenous components, the calixarene can also self-assemble, with numerous instances of macrocycle dimers.Calixarene complexation enables protein encapsulation, not merely side chain encapsulation. Cocrystal structures of sulfonato-calix[8]arene with cytochrome c or Ralstonia solanacearum lectin (RSL) provide evidence of encapsulation, with multiple calixarenes masking the same protein. NMR studies of cytochrome c and sulfonato-calix[8]arene are also consistent with multisite binding. In the case of RSL, a C₃ symmetric trimer, up to six calixarenes bind the protein yielding a cubic framework mediated by calixarene dimers. Biomolecular calixarene complexation has evolved from molecular recognition to framework construction. This latter development contributes to the challenge in design and preparation of porous molecular materials. Cytochrome c and sulfonato-calix[8]arene form frameworks with >60% solvent in which the degree of porosity depends on the protein:calixarene ratio and the crystallization conditions. Recent developments with RSL led to three frameworks with varying porosity depending on the crystallization conditions, particularly the pH. NMR studies indicate a pH-triggered assembly in which two acidic residues appear to play key roles. The field of supramolecular protein chemistry is growing, and this Account aims to encourage new developments at the interface between biomolecular and synthetic/supramolecular chemistry.

Figure 1

Schematic structures of (a) Gutsche’s canonical calix[4]arene with t-butyl groups on the upper rim and (b–h) water-soluble, protein-binding calixarenes. (b) Shinkai’s sulfonato-calix[n]arenes.⁻ (c) Representative glyco-calixarene, the tetra-galactoside from Parma. (d) Hamilton’s peptido-calixarene, containing glycine and aspartate. (e) Goto’s amphipathic calix[6]arene with lower rim carboxylates. (f) Schrader’s phosphonate-containing calix[4]arene. (g) de Mendoza’s guanidinio-calix[4]arene. (h) Hof’s asymmetric trisulfonato-calix[4]arene.

Figure 2

Binding site details from cocrystal structures of cytochrome c with (a) sulfonato-calix[4]arene, (b) phenyl-sulfonato-calix[4]arene, (c) methylphosphonato-calix[4]arene, (d) mono-PEGylated sulfonato-calix[4]arene, (e) sulfonato-thiacalix[4]arene with zinc, (f) phosphonato-calix[6]arene, (g) sulfonato-calix[6]arene, and (h) sulfonato-calix[8]arene. Lys4, shown as sticks, is encapsulated or bound exo depending on the calixarene.

Figure 3

(a) Filament of lysozyme tetramers (three shown) mediated by dimers of sulfonato-calix[4]arene and (b) detail of the calixarene dimer with encapsulation of Arg128 and a complex of Mg²⁺ and PEG. (c) Cocrystal structure of dimethylated lysozyme and sulfonato-calix[4]arene showing encapsulation of Lys116*.

Figure 4

NMR and X-ray data suggest autoregulated assembly of cytochrome c by sulfonato-calix[8]arene. ¹H–¹⁵N HSQC spectra of (a) pure protein and protein plus (b) ∼1 and (c) ∼3 equiv of calixarene. Spectral obliteration may be due to the formation of high molecular weight species such as a tetramer (based on PDB 6GD8). Higher equivalents of ligand result in masking/encapsulation (based on PDB 6GD9), disassembly, and spectral recovery.

Figure 5

Binding sites in cocrystal structures of PAF with (a) sulfonato-calix[4]arene, (b) sulfonato-calix[6]arene, and (c) sulfonato-calix[8]arene. Lys27, Pro29, Lys30, and Phe31 shown as sticks. Note PEG fragments in (b) and (c).

Figure 6

Binding sites and surface masking/encapsulation in three cocrystal structures of RSL with sulfonato-calix[8]arene, space groups (a) P2₁3, (b) I23 and (c) P3. The recurring epitope, Val13 and Lys34, shown as sticks. Note the disorder Lys34 in (a).

Figure 7

Porous frameworks of (a) cytochrome c (P4₃2₁2) and (b) RSL (I23) with unit cell axes a = b ≈ 10 nm. All interfaces are mediated by sulfonato-calix[8]arene.