Natural selective filtering systems (e.g., the extracellular matrix, nuclear pores, and mucus) separate molecules selectively and efficiently, and the detailed understanding of transport mechanisms exploited in these systems provides important bioinspired design principles for selective filters. In particular, nucleoporins consist of consensus repeat sequences that are readily utilized for engineering repeat proteins. Here, the consensus repeat sequence of Nsp1, a yeast nucleoporin, is polymerized to form a nucleoporin-like protein (NLP) and mutated to understand the effect of sequence on selective transport. The hydrophilic spacers of the NLPs were redesigned considering net charge, charge distribution, and polarity. Mutations were made near to and far from the FSFG interacting domain to explore the role of highly conserved residues as a function of spatial proximity. A nuclear transport receptor-cargo complex, nuclear transport factor 2-green fluorescent protein (NTF2-GFP), was used as a model for changes in transport. For mutations of the charged spacer, some mutations of highly conserved charged residues were possible without knocking out selective transport of the NTF2, but the formation of regions of clustered negative charge has an unfavorable effect on nuclear transporter permeation. Thus, positive net charge and alternating positive and negative charge within the hydrophilic spacer are advantageous for recognition and selective transport. In the polarity panel, mutations that increased the interaction between NTF2-GFP and the gel led to decreased permeation of the NTF2-GFP due to blocking of the interface and inability of the NTF2-GFP to transport into the gel. Therefore, these results provide a strategy for tuning selective permeability of biomolecules using the artificially designed consensus repeat-based hydrogels.

• PMID: 33428378
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Journal Article | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Yang YJ, Mai DJ, Li S, Morris MA, Olsen BD

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