Tuning Selective Transport of Biomolecules through Site-Mutated Nucleoporin-like Protein (NLP) Hydrogels
Tuning Selective Transport of Biomolecules through Site-Mutated Nucleoporin-like Protein (NLP) Hydrogels
复制标题
通过位点突变核孔蛋白样蛋白 (NLP) 水凝胶调节生物分子的选择性运输
DOI：
10.1021/acs.biomac.0c01083
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发表时间：
2021
影响因子：
6.2
通讯作者：
Olsen, Bradley D.
中科院分区：
文献类型：
--
作者：
Yang, Yun Jung;Mai, Danielle J.;Li, Shuaili;Morris, Melody A.;Olsen, Bradley D.
自然选择性过滤系统（例如，细胞外基质、核孔和粘液）选择性和有效地分离分子，并且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设计原理。特别地，核孔蛋白由易于用于工程化重复蛋白的共有重复序列组成。在这里，Nsp 1，酵母核孔蛋白的共识重复序列，聚合形成一个核孔蛋白样蛋白（NLP）和突变，了解选择性运输的序列的影响。考虑到净电荷、电荷分布和极性，重新设计了NLP的亲水间隔基。在FSFG相互作用结构域附近和远离FSFG相互作用结构域进行突变，以探索高度保守残基作为空间接近度的函数的作用。使用核转运受体-货物复合物，核转运因子2-绿色荧光蛋白（NTF 2-GFP）作为转运变化的模型。对于带电间隔区的突变，高度保守的带电残基的一些突变是可能的，而不会敲除NTF 2的选择性转运，但成簇负电荷区域的形成对核转运蛋白渗透有不利影响。因此，亲水性间隔物内的正净电荷和交替的正电荷和负电荷对于识别和选择性转运是有利的。在极性图中，增加NTF 2-GFP和凝胶之间相互作用的突变导致NTF 2-GFP的渗透降低，这是由于界面的阻断和NTF 2-GFP不能转运到凝胶中。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的选择性渗透性的策略。
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.