Site-Mutation of Nucleoporin Consensus Repeats for Understanding Selective Transport of Biomolecules

The nuclear membrane acts as a selective filter that regulates protein transport into the nucleus. The dense nucleoporin matrix rejects non-specific large molecules, and specific binding interactions between FG repeats in nucleoporin and nuclear transport factor (NTF2) allow the passage of specific large molecules. Previously, two nucleoporin-like proteins (NLPs) were designed to mimic selective separation property of nucleoporin. These NLP hydrogels specifically accumulate NTF2 in the hydrogel and allow its rapid penetration. Here, we perform systematic mutations to the NLP sequence to explore how the protein sequence affects transport properties. Mutants are specifically prepared to explore the role of highly conserved regions and electrostatic charge within the sequence. Transport of mutated NTF2 (NTF-W7A), which has lower affinity to FG repeats, is compared to NTF2 to understand the role of binding affinity on transport. Depending on the consensus sequences, the selectivity and transporting rates of NTF and NTF-W7A were changed. This finding gives us a way of tuning permeability and selectivity of biomolecules in the artificial protein gels for filtering and separation technologies.