Protein crystallization facilitated by molecularly imprinted polymers

Abstract

We present a new initiative and its application, namely the design of molecularly imprinted polymers (MIPs) for producing protein crystals which are essential for determining high-resolution 3-D 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 MIPs cavities and a protein-functionalised 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

Selective Crystallization of Proteins Using Engineered Nanonucleants

“…Very recently proteins to be crystallized were used as templates to imprint polymers, and these molecularly imprinted polymers were then used to crystallize a range of proteins. However, this work though interesting, does not provide a robust method which can be used to crystallize proteins reliably for a wide range of molecular weights, as during imprinting it is very difficult to control the aggregation and unfolding of the proteins 24 .…”

Structural Biology of Glycoproteins

“…Experience shows that by preparing glycoproteins with different glycoforms the chances of obtaining diffraction quality crystals are significantly increased. Combining this approach with the inclusion of novel additives in the crystallization experiment, for example "smart materials", such as the molecularly imprinted polymers (MIPs) recently reported by Sarkidakis et al [104], adds another dimension to crystallization.…”

Transferrin recognition based on a protein imprinted material prepared by hierarchical imprinting technique

“…During the past several years, the imprinting materials of macromolecules, especially proteins, were synthesized. Due to the high stability, low cost, and good selectivity to target macromolecules [12], they have been used in various fields, such as man-made enzyme inhibitors [13,14], solid phase separation [15,16], protein crystallization [17,18], and biosensors [19].…”