Making and Breaking Supramolecular Synthons for Modular Protein Frameworks.

Anionic calixarenes are useful mediators of protein assembly. In some cases, protein - calixarene cocrystallization yields multiple polymorphs. Ralstonia solanacearum lectin (RSL) cocrystallizes with p-sulfonato-calix[8]arene (sclx) in at least four distinct pH-dependent arrangements. One of these polymorphs, occurring at pH ≤ 4, is a cubic framework in which RSL nodes are connected by sclx dimers. These dimers are supramolecular synthons that occur in distinct crystal structures. Now, we show that the discus-shaped dimer of p-phosphonato-calix[6]arene (pclx), can replace the sclx dimer yielding a new assembly of RSL. Remarkably, just one type of RSL - pclx cocrystal was formed, irrespective of pH or crystallization condition. These results with pclx contrast starkly with sclx and suggest that the calixarene type (e.g., phosphonate versus sulfonate) dictates the synthon durability, which in turn exerts control over protein assembly and polymorph selection. Breaking the pclx dimer required a mutant of RSL with an affinity tag for macrocycle binding. This highly accessible, dicationic site resulted in a significantly altered and porous framework with pclx (but not with sclx). Experiments with ternary mixtures of RSL, pclx, and sclx provide evidence of pH-driven self-sorting. Thus, the "mix-and-match" of protein and supramolecular synthons is a promising approach to protein crystal engineering.