Precisely defined protein aggregates, as exemplified by crystals, have applications in functional materials. Consequently, engineered protein assembly is a rapidly growing field. Anionic calix[n]arenes are useful scaffolds that can mold to cationic proteins and induce oligomerization and assembly. Here, we describe protein-calixarene composites obtained via cocrystallization of commercially available sulfonato-calix[8]arene ( sclx 8 ) with the symmetric and “neutral” protein RSL. Cocrystallization occurred across a wide range of conditions and protein charge states, from pH 2.2–9.5, resulting in three crystal forms. Cationization of the protein surface at pH ∼ 4 drives calixarene complexation and yielded two types of porous frameworks with pore diameters >3 nm. Both types of framework provide evidence of protein encapsulation by the calixarene. Calixarene-masked proteins act as nodes within the frameworks, displaying octahedral-type coordination in one case. The other framework formed millimeter-scale crystals within hours, without the need for precipitants or specialized equipment. NMR experiments revealed macrocycle - modulated side chain p K a values and suggested a mechanism for pH-triggered assembly. The same low pH framework was generated at high pH with a permanently cationic arginine-enriched RSL variant. Finally, in addition to protein framework fabrication, sclx 8 enables de novo structure determination.

Additional Q7 binding sites drive protein aggregation in solution and statistical disorder in the crystalline biohybrid suggest new possibilities for protein-based materials.

Controlled protein assembly is an enabling technology, for example, in the bottom-up fabrication of biomaterials. This paper describes the assembly of a β-propeller protein using two orthogonal interaction modes. Previously, protein assembly was directed by metal coordination or macrocycle complexation. Here, we demonstrate the combination of metal coordination and macrocycle complexation for controlled assembly. An established protein−cucurbit[7]uril (Q7) assembly, which relies on trimeric Q7 clusters, was modified by the inclusion of metal-binding sites in the protein. The application of zinc−histidine coordination to tune the Q7-induced assembly resulted in metallo-bioorganic crystalline architectures. The relative arrangement of the protein−Q7 layers was reorganized by the zinc bridging ions. One structure resulted in a different type of protein−Q7 packing that involves Q7 dimers. Apparently, Q7 is a versatile molecular glue that can be combined with metal-mediated protein assembly. This dual strategy expands significantly the toolkit for engineered assembly.

Highlights d KIF18B, a nuclear kinesin, is required for efficient end-joining of DSBs d A Tudor-interacting motif (TIM) in KIF18B directly interacts with the 53BP1 Tudor d KIF18B's TIM facilitates the binding of 53BP1 to chromatin in the vicinity of DSBs d The motor activity KIF18B is also required for efficient 53BP1 recruitment

Controlled protein assembly and crystallization is necessary as a means of generating diffraction-quality crystals as well as providing a basis for new types of biomaterials. Water-soluble calixarenes are useful mediators of protein crystallization. Recently, it was demonstrated that Ralstonia solanacearum lectin (RSL) co-crystallizes with anionic sulfonato-calix[8]arene (sclx8) in three space groups. Two of these co-crystals only grow at pH ≤ 4 where the protein is cationic, and the crystal packing is dominated by the calixarene. This paper describes a fourth RSL–sclx8 co-crystal, which was discovered while working with a cation-enriched mutant. Crystal form IV grows at high ionic strength in the pH range 5–6. While possessing some features in common with the previous forms, the new structure reveals alternative calixarene binding modes. The occurrence of C 2-symmetric assemblies, with the calixarene at special positions, appears to be an important result for framework fabrication. Questions arise regarding crystal screening and exhaustive searching for polymorphs.

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.

One approach to protein assembly involves watersoluble supramolecular receptors that act like glues. Bionanoarchitectures directed by these scaffolds are often systemspecific, 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 β-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 intrinsi-cally 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.

PEGylation is the most widely used half-life extension strategy for protein therapeutics. While it imparts a range of attractive attributes PEGylation can impede protein binding and reduce efficacy. A model system to probe the effects of PEGylation on protein binding has practical applications. Here, we present a system based on complex formation between a hexavalent lectin (RSL) and the globular polysaccharide Ficoll PM70 (a type of glycocluster). Mutants of the lectin were used to generate conjugates with 3, 6, or 12 PEG (1 kDa) chains. Using NMR spectroscopy we monitored how the degree of PEGylation impacted the lectin–Ficoll interaction. The binding propensity was observed to decrease with increasing polymer density. Apparently, the extended PEG chains sterically impede the lectin–Ficoll binding. This deduction was supported by molecular dynamics simulations of the protein–polymer conjugates. The implications for protein–surface interactions are discussed.