Protein Frameworks with Thiacalixarene and Zinc.

Controlled protein assembly provides a means to generate biomaterials. Synthetic macrocycles such as the water-soluble sulfonato-calix[n]arenes are useful mediators of protein assembly. Sulfonato-thiacalix[4]arene (tsclx 4 ), with its metal-binding capacity, affords the potential for simultaneous macrocycle- and metal-mediated protein assembly. Here, we describe the tsclx 4 -/Zn-directed assembly of two proteins: cationic α-helical cytochrome c (cyt c) and neutral ß-propeller Ralstonia solanacearum lectin (RSL). Two co-crystal forms were obtained with cyt c, each involving multinuclear zinc sites supported by the cone conformation of tsclx 4 . The tsclx 4 /Zn cluster acted as an assembly node via both lysine encapsulation and metal-mediated protein-protein contacts. In the case of RSL, tsclx 4 adopted the 1,2-alternate conformation and supported a dinuclear zinc site with concomitant encapsulation and metal-binding of two histidine side chains. These results, together with the knowledge of thiacalixarene/metal nanoclusters, suggest promising applications for thiacalixarenes in biomaterials and MOF fabrication.

Segregated Protein-Cucurbit[7]uril Crystalline Architectures via Modulatory Peptide Tectons.

One approach to protein assembly involves water-soluble supramolecular receptors that act like glues. Bionanoarchitectures directed by these scaffolds are often system-specific, with few studies investigating their customization. Herein, the modulation of cucurbituril-mediated protein assemblies through the inclusion of peptide tectons is described. Three peptides of varying length and structural order were N-terminally appended to RSL, a ß-propeller building block. Each fusion protein was incorporated into crystalline architectures mediated by cucurbit[7]uril (Q7). A trimeric coiled-coil served as a spacer within a Q7-directed sheet assembly of RSL, giving rise to a layered material of varying porosity. Within the spacer layers, the coiled-coils were dynamic. This result prompted consideration of intrinsically disordered peptides (IDPs) as modulatory tectons. Similar to the coiled-coil, a mussel adhesion peptide (Mefp) also acted as a spacer between protein-Q7 sheets. In contrast, the fusion of a nucleoporin peptide (Nup) to RSL did not recapitulate the sheet assembly. Instead, a Q7-directed cage was adopted, within which disordered Nup peptides were partially "captured" by Q7 receptors. IDP capture occurred by macrocycle recognition of an intrapeptide Phe-Gly motif in which the benzyl group was encapsulated by Q7. The modularity of these protein-cucurbituril architectures adds a new dimension to macrocycle-mediated protein assembly. Segregated protein crystals, with alternating layers of high and low porosity, could provide a basis for new types of materials.

Noncovalent Protein-Pseudorotaxane Assembly Incorporating an Extended Arm Calix[8]arene with α-Helical Recognition Properties.

Water-soluble, anionic calix[n]arenes are useful receptors for protein recognition and assembly. For example, sulfonato-calix[8]arene (sclx 8 ) can encapsulate proteins and direct their assembly into porous frameworks. In this work, we turned our attention to an "extended arm" calixarene with 16 phenyl rings. We hypothesized that this larger receptor would have increased capacity for protein masking/encapsulation. A cocrystal structure of p-benzyl-sulfonato-calix[8]arene (b-sclx 8 ) and cytochrome c (cyt c) revealed a surprising assembly. A pseudorotaxane comprising a stack of three b-sclx 8 molecules threaded by polyethylene glycol (PEG) was bound to the protein. The trimeric b-sclx 8 stack, a tubelike structure with a highly charged surface, mediated assembly via a new mode of protein recognition. The calixarene stack presents four hydrophobic grooves, each of which binds to one cyt c by accommodating the N-terminal α-helix. This unprecedented binding mode suggests new possibilities for supramolecular protein chemistry.

Porous assembly of an antifungal protein mediated by zinc and sulfonato-calix[8]arene.

Controlled protein assembly holds great potential in the fabrication of biohybrid materials. However, the structural diversity and complexity of proteins present an obstacle to controlled assembly. Supramolecular chemistry is a possible solution as it offers tools to mediate self-assembly with molecular precision. This paper deals with the calixarene- and zinc-mediated assembly and crystallization of the histidine-rich Penicillium chrysogenum antifungal protein B (PAFB). We report crystal structures of pure PAFB, PAFB in complex with Zn2+, and the ternary complex of PAFB, Zn2+ and sulfonato-calix[8]arene (sclx8). A comparison of the three crystal structures revealed the structural plasticity of PAFB. While the flexible and highly anionic sclx8 resulted in large molecular weight aggregates of PAFB in solution, diffraction-quality crystals of PAFB-sclx8 were not obtained. We report crystals of PAFB-Zn2+-sclx8 in which a trinuclear zinc cluster occurred adjacent to a calixarene binding site. Interestingly, the combination of sclx8 complexation and zinc coordination resulted in a porous framework with channels of circa 2 nm diameter. Detailed analysis of the crystal structure highlighted novel molecular recognition features. This research enriches the set of supramolecular interactions available to promote protein assembly.

Protein recognition by cucurbit[6]uril: high affinity N-terminal complexation.

The donut-shaped cucurbit[n]urils (Qn, n = 6-8) are rigid macrocyclic receptors with widespread use in protein recognition. To date, most applications have centred on the encapsulation of N-terminal aromatic residues by Q7 or Q8. Less attention has been placed on Q6, which can recognize lysine side chains due to its high affinity for alkylamines. In this work, we investigated protein-Q6 complexation by using NMR spectroscopy. Attempts to crystallize protein-Q6 complexes were thwarted by the crystallization of Q6. We studied four proteins that vary in size, net charge, and lysine content. In addition to Q6 interactions with specific Lys or dimethylated Lys residues, we report striking evidence for N-terminal recognition. High affinity (micromolar) binding occurred with the N-terminal Met-Lys motif present in one of the four model proteins. Engineering this feature into another model protein yielded a similar high affinity site. We also present evidence for Q8 binding at this N-terminal feature. These data expand the cucurbituril toolkit for protein sensing.

Multidentate, V-Shaped Pyridine Building Blocks as Tectons for Crystal Engineering.

The formation of supramolecular structural units through self-assembly is a powerful method to design new architectures and materials endowed with specific properties. With the aim of adding a group of versatile tectons to the toolkit of crystal engineers, we have devised and synthesised four new V-shaped building blocks characterised by an aryl acetylene scaffold comprising three substituted pyridine rings connected by two triple bonds. The judicious choice of different substituents on the pyridine rings provides these tectons with distinctive steric, electrostatic and self-assembly properties, which influence their crystal structures and their ability to form co-crystals. Co-crystals of the tectons with tetraiododifluorobenzene were obtained both via traditional and mechanochemical crystallisation strategies, proving their potential use in crystal engineering. The energetic contributions of the supramolecular interactions at play in the crystal lattice have also been evaluated to better understand their nature and strength and to rationalise their role in designing molecular crystals.

Mechanically-Driven Vase-Kite Conformational Switch in Cavitand Cross-Linked Polyurethanes.

The eligibility of tetraquinoxaline cavitands (QxCav) as molecular grippers relies on their unique conformational mobility between a closed (vase) and an open (kite) form, triggered in solution by conventional stimuli like pH, temperature and ion concentration. In the present paper, the mechanochemical conformational switching of ad hoc functionalized QxCav covalently embedded in an elastomeric polydimethylsiloxane and in a more rigid polyurethane matrix is investigated. The rigid polymer matrix is more effective in converting mechanical force into a conformational switch at the molecular level, provided that all four quinoxaline wings are covalently connected to the polymer.

Engineered assembly of a protein-cucurbituril biohybrid.

A crystalline biohybrid with a 4 : 1 protein : cucurbituril mass ratio is presented. This result was achieved by engineering additional cucurbit[7]uril (Q7) binding sites into a ß-propeller protein. In contrast to the parent protein, Q7-controlled assembly of the engineered variant occurred in solution, as evidenced by NMR and SAXS measurements.

Solvent-responsive cavitand lanthanum complex.

Stimuli-responsive supramolecular assemblies are dynamic systems that can reversibly switch between different states upon external stimuli. In this context, metal coordination offers a reliable strategy for the preparation of stimuli responsive supramolecular architectures. Herein we report the preparation of a solvent-responsive cavitand-lanthanum coordination complex. A tetra-phosphonate cavitand has been functionalized with four hydroxyl moieties at the upper rim to form a pre-organized octadentate ligand capable of binding lanthanum salts. Exploiting the orthogonal recognition sites, two different complex architectures are formed in acetonitrile and acetone, respectively. The complexes have been characterized in solution by NMR spectroscopy, ITC experiments, while at the solid state, the single crystal structure of the acetonitrile derivative has been determined. Furthermore, as observed by DOSY-NMR spectroscopy, small quantities of acetone in acetonitrile are sufficient to trigger assembly interconversion.

Cucurbit[7]uril-Dimethyllysine Recognition in a Model Protein.

Here, we provide the first structural characterization of host-guest complexation between cucurbit[7]uril (Q7) and dimethyllysine (KMe2 ) in a model protein. Binding was dominated by complete encapsulation of the dimethylammonium functional group. While selectivity for the most sterically accessible dimethyllysine was observed both in solution and in the solid state, three different modes of Q7-KMe2 complexation were revealed by X-ray crystallography. The crystal structures revealed also entrapped water molecules that solvated the ammonium group within the Q7 cavity. Remarkable Q7-protein assemblies, including inter-locked octahedral cages that comprise 24 protein trimers, occurred in the solid state. Cucurbituril clusters appear to be responsible for these assemblies, suggesting a strategy to generate controlled protein architectures.