We present a previously undescribed initiative and its application, namely the design of molecularly imprinted polymers (MIPs) for producing protein crystals that are essential for determining high-resolution 3D structures of proteins. MIPs, also referred to as “smart materials,” are made to contain cavities capable of rebinding protein; thus the fingerprint of the protein created on the polymer allows it to serve as an ideal template for crystal formation. We have shown that six different MIPs induced crystallization of nine proteins, yielding crystals in conditions that do not give crystals otherwise. The incorporation of MIPs in screening experiments gave rise to crystalline hits in 8–10% of the trials for three target proteins. These hits would have been missed using other known nucleants. MIPs also facilitated the formation of large single crystals at metastable conditions for seven proteins. Moreover, the presence of MIPs has led to faster formation of crystals in all cases where crystals would appear eventually and to major improvement in diffraction in some cases. The MIPs were effective for their cognate proteins and also for other proteins, with size compatibility being a likely criterion for efficacy. Atomic force microscopy (AFM) measurements demonstrated specific affinity between the MIP cavities and a protein-functionalized AFM tip, corroborating our hypothesis that due to the recognition of proteins by the cavities, MIPs can act as nucleation-inducing substrates (nucleants) by harnessing the proteins themselves as templates.

We present a theory and experiments that help clarify the origin of the effectiveness of nanoporous substrates in the heterogeneous nucleation of protein crystals. The central idea tested here is that when a substrate (or “nucleant”) possesses pores of the order of the hydrodynamical radius of a protein, then the entropic penalty associated with nucleating a protein crystal on that surface may be alleviated. Model experiments using lysozyme and nanoporous gold (NPG) substrates suggest that there is indeed a reduction in the entropy associated with creating critical nuclei, but the magnitude of the reduction is small. Taken together with further examination of protein crystallization with NPG nucleants using four other proteins, our aggregate results suggest that surface chemistry and surface area effects play the dominant role in nucleation when using these nanoporous nucleants.