Supramolecular Synthons in Protein-Ligand Frameworks.

Supramolecular synthons, defined as reproducible intermolecular structural units, have greatly aided small molecule crystal engineering. In this paper, we propose that supramolecular synthons guide ligand-mediated protein crystallization. The protein RSL and the macrocycle sulfonato-calix[8]arene cocrystallize in at least four ways. One of these cocrystals is a highly porous cube comprising protein nodes connected by calixarene dimers. We show that mutating an aspartic acid to an asparagine results in two new cubic assemblies that depend also on the crystallization method. One of the new cubic arrangements is mediated by calixarene trimers and has a ∼30% increased cell volume relative to the original crystal with calixarene dimers. Crystals of the sulfonato-calix[8]arene sodium salt were obtained from buffered conditions similar to those used to grow the protein-calix[8]arene cocrystals. X-ray analysis reveals a coordination polymer of the anionic calix[8]arene and sodium cation in which the macrocycle is arranged as staggered stacks of the pleated loop conformation. Remarkably, the calixarene packing arrangement is the same in the simple salt as in the protein cocrystal. With the pleated loop conformation, the calixarene presents an extended surface for binding other calixarenes (oligomerization) as well as binding to a protein patch (biomolecular complexation). Small-angle X-ray scattering data suggest pH-dependent calixarene assembly in solution. Therefore, the calix[8]arene-calix[8]arene structural unit may be regarded as a supramolecular synthon that directs at least two types of protein assembly, suggesting applications in protein crystal engineering.

Multivalent Calixarene Complexation of a Designed Pentameric Lectin.

We describe complex formation between a designed pentameric ß-propeller and the anionic macrocycle sulfonato-calix[8]arene (sclx8), as characterized by X-ray crystallography and NMR spectroscopy. Two crystal structures and 15N HSQC experiments reveal a single calixarene binding site in the concave pocket of the ß-propeller toroid. Despite the symmetry mismatch between the pentameric protein and the octameric macrocycle, they form a high affinity multivalent complex, with the largest protein-calixarene interface observed to date. This system provides a platform for investigating multivalency.

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.