Protein crystallization facilitated by molecularly imprinted polymers

Abstract

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.

Fig. 1.

MIF crystallization trials in the presence of T-MIP and NIP. The MIP and NIP have a translucent gel-like appearance. When added to crystallization drops, they spread out and can fragment. (A) A single MIF crystal grown in a drop containing T-MIP. The MIP is indicated by the arrow. Scale bar corresponds to 0.1 mm. (B) Drop containing NIP at identical conditions; no crystals are formed. Scale bar corresponds to 0.15 mm.

Fig. 2.

Progression of the formation of trypsin crystals on trypsin-imprinted MIP. (A) Phase separation, (B) crystalline aggregation at the protein-rich droplets (Bottom Left), and large single crystal. Scale bars correspond to 0.05 mm.

Fig. 3.

Results of screening with the Index screen. (A) A hit containing MIF crystals in solution 5, scale bar corresponds to 0.15 mm. (B) A hit containing alpha crustacyanin crystals in solution 43, scale bar corresponds to 0.05 mm. The color of the alpha crustacyanin protein is blue, hence the dark color of the crystals.

Fig. 4.

A typical force-distance graph detailing the interrogation of MIP with a BHb-conjugated 10 nm (radius of curvature) silicon nitride AFM probe. The gray line shows the tip descending, initially without contact with the surface. At some point, the tip jumps into contact with the surface and indents into it. The black line shows the tip retracting: The adhesion/bonding between tip and sample causes the cantilever to adhere to the sample. As the retraction continues, the adhesion breaks. The cycle can then be repeated.