Making and Breaking Supramolecular Synthons for Modular Protein Frameworks

Abstract 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 8 ) 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 8 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 6 ), can replace the sclx 8 dimer yielding a new assembly of RSL. Remarkably, just one type of RSL – pclx 6 cocrystal was formed, irrespective of pH or crystallization condition. These results with pclx 6 contrast starkly with sclx 8 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 6 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 6 (but not with sclx 8 ). Experiments with ternary mixtures of RSL, pclx 6 , and sclx 8 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.