Abstract
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 (sclx8) 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 pKa 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, sclx8 enables de novo structure determination.
Highlights
Protein-based materials have great potential to serve society.[1−4] With their periodic arrangement of functional building blocks, crystals have applications in catalytic devices.[3−7] Porous crystals are of particular interest considering their capacity to capture and transform biomolecules.[6,8−14] While great advances have been achieved with metal organic frameworks (MOFs)[12] and covalent organic frameworks (COFs),[13] protein-based frameworks have proved more challenging.[11,14−17] Yet, biocompatible and biodegradable frameworks are highly desirable given the demands for new therapeutics and biomaterials as well as sustainable manufacturing processes.[2]
Various sophisticated frameworks have been described to date, usually with the requirement for protein engineering or specific assembly inducing ligands.[19,23,25−27,47−49] Sulfonato-calixarenes are commercially available: biocompatible ligands that can be combined with protein building blocks to yield frameworks in a manner reminiscent of MOF manufacture using off-the-shelf reagents.[12]
The data presented here greatly widen the scope for sclxn-assisted protein assembly and crystallization to include “neutral” target proteins
Summary
Protein-based materials have great potential to serve society.[1−4] With their periodic arrangement of functional building blocks, crystals have applications in catalytic devices.[3−7] Porous crystals are of particular interest considering their capacity to capture (store) and transform biomolecules.[6,8−14] While great advances have been achieved with metal organic frameworks (MOFs)[12] and covalent organic frameworks (COFs),[13] protein-based frameworks have proved more challenging.[11,14−17] Yet, biocompatible and biodegradable frameworks are highly desirable given the demands for new therapeutics and biomaterials as well as sustainable manufacturing processes.[2]. Article revealed binding patches consistent with the crystallographically defined protein-calixarene interfaces (Figure 5C) The occurrence of both binding sites (as per crystal form III) in the NMR experiments suggests transient sharing of bound sclx[8] between two RSL trimers. The versatility of guanidinium groups at protein−protein and protein−ligand interfaces is well-established.[53,82−84] We reason that the “stickiness” of the Arg-rich variants toward sclx[8] yielded highly encapsulated protein-calixarene particles incapable of self-association This hypothesis is supported by cocrystallization of RSL-R6 and RSL-R8 occurring only at high salt concentration wherein counterions compete for sclx[8] complexation and lower its affinity for the protein surface. The different pore dimensions in the frameworks (Table 2) along with the varying solvent accessibilities of the calixarene (∼35, 45 and 55% solvent exposed in crystal forms I, II and III, respectively, Figure 1 and Table S5) result in materials with different ligand uptake capacities
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