Tuning Selective Transport of Biomolecules through Site-Mutated Nucleoporin-like Protein (NLP) Hydrogels,Biomacromolecules

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Tuning Selective Transport of Biomolecules through Site-Mutated Nucleoporin-like Protein (NLP) Hydrogels
Biomacromolecules ( IF 5.5 ) Pub Date : 2021-01-11 , DOI: 10.1021/acs.biomac.0c01083
Yun Jung Yang _{1,

2} , Danielle J Mai _{1,

3} , Shuaili Li ₁ , Melody A Morris ₁ , Bradley D Olsen ₁

Affiliation

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.

中文翻译：

通过定点突变的核蛋白样蛋白（NLP）水凝胶调节生物分子的选择性运输。

天然的选择性过滤系统（例如，细胞外基质，核孔和粘液）选择性地和有效地分离分子，对这些系统中利用的转运机制的详细理解为选择性过滤器提供了重要的生物启发性设计原理。特别地，核孔蛋白由容易用于工程化重复蛋白的共有重复序列组成。此处，酵母核孔蛋白Nsp1的共有重复序列被聚合形成核孔蛋白样蛋白（NLP），并进行突变以了解该序列对选择性转运的影响。考虑到净电荷，电荷分布和极性，对NLP的亲水性间隔基进行了重新设计。在接近和远离FSFG相互作用域的地方进行突变，以探索高度保守的残基作为空间邻近度的作用。核转运受体-货物复合物，核转运因子2-绿色荧光蛋白（NTF2-GFP），被用作转运变化的模型。对于带电间隔基的突变，在不敲除NTF2选择性转运的情况下，可以对高度保守的带电残基进行某些突变，但是簇状负电荷区域的形成对核转运蛋白的渗透具有不利影响。因此，亲水性间隔基内的正净电荷以及正负电荷交替对于识别和选择性转运是有利的。在极性面板中，突变增加了NTF2-GFP与凝胶之间的相互作用，导致NTF2-GFP的渗透性降低，这是由于界面的封闭和NTF2-GFP无法转运到凝胶中所致。因此，这些结果提供了使用人工设计的基于共有重复序列的水凝胶调节生物分子选择性渗透性的策略。

更新日期：2021-02-08

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