(55c) Engineering Selective Biomolecular Transport in Nucleoporin-like Protein Hydrogels

Engineering new materials that control specific biomolecular transport will be central to numerous technologies including biosensors, bioseparations, and tissue engineering. Nuclear pore complexes naturally regulate biomolecular transport with remarkable specificity (>99.9% of proteins rejected) and speed (1,000 proteins per second per pore). The central channel of the nuclear pore is filled with a disordered matrix of nucleoporin proteins responsible for recognition and selective transport of nuclear transport receptors (NTRs).

Recently, artificially engineered nucleoporin-like polypeptides (NLPs) replicated the selective permeability of NTRs observed in natural nucleoporin proteins. NLPs consist of associative domains that promote gelation and formation of hydrogel structure, as well as affinity domains based on minimal consensus repeat sequences from the yeast nucleoporin Nsp1. We present the biophysical characterization of NLPs using small-angle neutron scattering, fluorimetric transport assays, and NTR binding assays, which reveal the importance of entropic size exclusion and moderate binding affinity on selective permeability in NLP hydrogels. NLP affinity domains are also modified to tune transport properties in a family of NLP mutants. Here, amino acid site mutations that vary charge, polarity, and hydrophobicity suggest the importance of highly conserved residues on selective transport and affinity to NTRs. Overall, NLPs provide a bioinspired platform that enables systematic studies of sequence–property relationships in selectively permeable protein hydrogels.